Methods, devices, arrays and kits for detecting and analyzing biomolecules

ABSTRACT

The present disclosure is directed to devices, arrays, kits and methods for detecting biomolecules in a tissue section (such as a fresh or archival sample, tissue microarray, or cells harvested by an LCM procedure) or other substantially two-dimensional sample (such as an electrophoretic gel or cDNA microarray) by creating “carbon copies” of the biomolecules eluted from the sample and visualizing the biomolecules on the copies using one or more detector molecules (e.g., antibodies or DNA probes) having specific affinity for the biomolecules of interest. Specific methods are provided for identifying the pattern of biomolecules (e.g., proteins and nucleic acids) in the samples. Other specific methods are provided for the identification and analysis of proteins and other biological molecules produced by cells and/or tissue, especially human cells and/or tissue. The disclosure also provides a plurality of differentially prepared and/or processed membranes that can be used in described methods, and which permit the identification and analysis of biomolecules.

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Continuation in Part (CIP) of U.S. patentapplication Ser. No. 09/753,574 (filed Jan. 4, 2001), which is a CIP ofU.S. patent application Ser. No. 09/718,990 (Filed Nov. 20, 2000), whichis a CIP of International Patent Application No. US00/20354, filed Jul.26, 2000 and published in the English language, and claims the benefitof U.S. Provisional Patent Application No. 60/145,613 (filed Jul. 26,1999). The current application further claims the benefit of U.S.Provisional Patent Application Nos. 60/286,258 (filed Apr. 25, 2001),60/304,031 (filed Jul. 9, 2001), and 60/296,475 (filed Jun. 8, 2001).Each of these related applications is incorporated herein in theirentirety.

STATEMENT OF GOVERNMENT RIGHTS

[0002] At least one of the inventors is an employee of an agency of theGovernment of the United States, and the Government may have certainrights in this invention.

FIELD OF THE DISCLOSURE

[0003] The present disclosure is directed to methods, devices, arrays,and kits for identifying and analyzing large numbers of biomolecules ina sample, such as a biological sample. The disclosure further relates tousing these methods, devices, arrays, and kits to help determine thefunction and role of biomolecules in disease, and to correlating thepresence, absence, or quantity of a combination of biomolecules withparticular diseases, prognoses, or responses to therapies.

BACKGROUND OF THE DISCLOSURE

[0004] Now that the 50,000 or so genes that make up the human genomehave been sequenced, tools are needed to determine when and in what typeof tissue those genes are active so as to ascertain their function androle, particularly in disease. This effort, often referred to as“functional genomics” and “proteomics,” is especially important inefforts to discover new drugs since new pharmaceutical agents are beingdesigned to target specific enzymes, receptors, and other proteins.Eventually, proteomic information will be used in clinical diagnosticsto help guide treatment selection in the emerging era of “personalizedmedicine.”

[0005] Some believe that the 100,000 human genes may turn out to produceup to a million different protein variants due to post-translational andother modifications. Within the next decade the pharmaceutical industryis expected to identify up to 10,000 proteins against which humantherapeutics can be directed. Additional therapeutics, gene modifiers,expression modifiers, and valuable biomolecules also are expected to bedeveloped or identified through the extension of proteomics to theanalysis of non-human animals and plants.

[0006] Although there may be up to a million different protein variantsin humans, only about 10,000-15,000 proteins are expressed in anyparticular cell type. Thus, for example, liver cells have essentiallythe same genome as skin cells taken from the same individual, but thetwo cell populations express substantially different sets of proteins.It is often desirable, therefore, to profile and compare the patterns ofproteins (i.e., the “proteome” of a cell) in different cell populations(e.g., diseased and normal tissue; fetal and mature tissue; human andnon-human tissue, etc.) to identify targets for drugs.

[0007] One common approach to establishing or confirming the associationof gene activity with disease is through expression analysis. DNAmicroarrays are used to survey differential expression patterns ofthousands of genes from extracts taken from samples of tissuesrepresenting various diseases. If particular genes are expressed indiseased tissue but not in normal tissue they may be relevant asdiagnostic markers and targets of pharmaceutical intervention. Onedisadvantage with this approach is that the sample being tested isdisassociated from the tissue from which it was isolated, thereby losingthe ability to observe gene expression patterns in the context of thetissue in which the genes are active. Since the morphologicalrelationship is not preserved in microarray analysis, it is hard to knowwhat component of the sample is responsible for the changes observed ingene expression. Also, microarray analysis is usually performed on ahomogenized sample of tissue, making it virtually impossible to ascribeexpression to a specific cell type, let alone a specific cell.

[0008] In situ detection and visualization of proteins traditionally hasbeen accomplished through immuno-histochemistry (IHC). This techniqueinvolves the mounting a thin tissue section on the glass slide andvisualizing a protein of interest with a detectable antibody that hasspecific binding affinity for the target protein. Because of certaintechnical limitations of IHC, only one or two proteins from a singletissue section can be achieved. Also, proteins are still embedded in thetissue and are not presented to the antibodies in the most appropriateway (proteins are not highly denatured) lowering the success rate of theantibody reactivity.

[0009] The most widely used method for identifying and measuringproteins and nucleic acids that have been removed from tissue samples isgel electrophoresis. Electrophoresis generally refers to techniques forseparating or resolving molecules in a mixture under the influence of anapplied electric field. Separation is based on difference in (usually)the size and/or charge of the molecules. Molecules separated byelectrophoresis are often visualized by staining with a non-specificdye, such as Coomassie blue (for proteins) or ethidium bromide (fornucleic acids). Such dye staining does not specifically identifyindividual molecules. Furthermore, ubiquitous dye staining is generallynot very sensitive.

[0010] More sensitive detection methods exist, such as antibody-baseddetection for proteins. In particular, immunoblotting, also known as“Western blotting,” is often used to detect gel-separated proteins. Thistechnique uses detectable antibodies specific to the proteins ofinterest in lieu of a ubiquitous stain. A key imitation of the techniqueis its low throughput; at most only a handful of proteins can beidentified from a single lane of an immunoblot on a single blot, due tooverlapping banding patterns and cross reactivity of antibodies withdifferent proteins in the sample. Thus, immunoblotting is typicallyperformed using only one antibody per membrane to ensure specificity.

[0011] Though it is possible to strip and re-probe an immunoblot,stripping will also remove protein of the sample that had been bound tothe membrane, thus encumbering quantitative analysis of the sample.Moreover, the proportion of each individual protein removed from themembrane by such treatment will vary depending upon the nature of theprotein, which further clouds efforts to quantitate the relative amountsof protein initially present in the sample. There remains a clear needto develop blotting techniques that permit larger numbers of antigens tobe detected simultaneously from a single test sample.

[0012] It would be desirable to have high-throughput approaches fordetecting, identifying and comparing large numbers of biomarkers that isrelatively inexpensive, can be used by ordinary laboratory personnel,and readily permits the capture, organization, and analysis of the datagenerated thereby.

SUMMARY OF THE DISCLOSURE

[0013] The present disclosure is directed to devices and methods fordetecting biomolecules in a substantially two-dimensional sample (e.g.tissue section, tissue array, electrophoretic gel, and so forth) bycreating substantial copies of the biomolecules eluted from the sample.The biomolecules then can be visualized on the copies using detectors,for example antibodies or DNA probes, having specific affinity for thebiomolecules of interest.

[0014] The present disclosure is further directed to methods and devicesfor identifying the pattern of biomolecules (e.g., proteins and nucleicacids) expressed in tissue samples, and for correlating the expressionpattern with, for instance, various diseases, prognoses, or responses totherapies.

[0015] Provided herein are methods of detecting biomolecules in asample, which methods involve providing a stack of at least two layeredmembranes; applying the sample to the stack under conditions that permitmovement of the biomolecules through multiple layered membranes of thestack, and allow capture of at least a portion of the biomolecules onthe membranes, and detecting the biomolecules on one or more of themultiple membranes. In specific examples of such methods, thebiomolecules are captured directly by the membranes. Certain membranesfor use in such methods have a high affinity but low capacity forbiomolecules, for instances proteins, nucleic acids, lipids,carbohydrates, or combinations thereof.

[0016] Another embodiment of the disclosure is a method of makingmultiple substantial copies (which need not be identical) of abiological sample. These methods involve providing a stack of layeredmembranes, wherein the membranes permit biomolecules applied to thestack to move through a plurality of the membranes, while capturing (forinstance, directly) at least a portion of the biomolecules on multiplemembranes and applying the biological sample to the stack, underconditions that allow the multiple membranes to capture at least aportion of the biomolecules from the sample. This forms the multiplesubstantial copies of the biological sample.

[0017] Samples for use in examples of provided methods are (or can bemade) substantially two-dimensional; representative non-limiting typesof samples include tissue sections, tissue microarrays, tissuemacroarrays, laser capture microdissected tissue samples, andelectrophoretic gels (e.g., 1-D or 2-D electrophoretic gels).

[0018] Yet a further embodiment is a method of creating a set ofmicroarray copies, which method involves providing a stack of layeredmembranes, and applying a plurality of molecules (e.g. DNA probes,antibodies, or a combination thereof), to the stack of layeredmembranes. In examples of such methods, the stack of layered membranesincludes a plurality of substrates through which the molecules move, andin which a portion of the molecules are directly captured by one or moreof the substrates.

[0019] Another specific embodiment is a method of analyzing biomoleculesin a tissue sample, which method involves providing at least onemembrane (in some embodiments, a plurality of membranes), positioningthe at least one (or more) membrane in contact with the tissue sample,and applying heat and/or pressure to the tissue sample, whereuponbiomolecules are transferred from the tissue sample onto the at leastone membrane (referred to generally as contact transfer). One or more ofthe biomolecules can then be analyzed on the at least one membrane.

[0020] Another example of a provided method is a method of replicatingbiomolecular content of a tissue array (such as a micro- or macroarray),which method involves providing the tissue array and transferringbiomolecules from the tissue array onto a plurality of membranes so asto produce at least one replicate of the biomolecular content of thetissue array.

[0021] The disclosure also provides a method of analyzing cellularmaterial embedded on an LCM transfer film, which method involvesproviding one or more membranes, positioning the one or more membranesadjacent to the LCM transfer film, transferring biomolecules from thecellular material to the one or more membranes, and detecting thebiomolecules on the membranes.

[0022] Further encompassed methods include methods for analyzing theproteome of a biological sample. Examples of such methods involveseparating at least one protein from another protein present in thebiological sample, transferring a portion of the separated protein to aplurality of membranes in a stacked configuration, incubating each ofthe membranes in the presence of one or more different species ofpredetermined ligand molecules (or detector molecules) under conditionssufficient to permit binding between the separated protein and aligand/detector capable of binding to such protein; and analyzing theproteome by determining the occurrence of binding between the proteinand any of the species of predetermined ligand molecules.

[0023] A further embodiment is a method for identifying biomoleculesthat have been separated on a solid support (e.g., a 1-D or 2-D gel).Such methods involve providing a solid support containing the separatedbiomolecules, wherein the support has an upper side and a lower side,applying a first stack of membranes to the upper side and a second stackof membranes to the lower side, permitting the biomolecules to betransferred from the support to the first and second membrane stacks,and separating the membranes. The transferred biomolecules can then bedetected, identified, or otherwise analyzed on at least one of themembranes.

[0024] The disclosure also provides kits. Examples of kits include amembrane array for detecting biomolecules in a sample, and one or morecontainers of detector molecules for detecting molecules captured onmembranes of the array. Arrays included in such kits contain a pluralityof membranes, each of which has substantially a same affinity forbiomolecules that may be analyzed using the kit.

[0025] Another kit embodiment is a kit for comparing the molecularprofiles of tissue samples. Such kits contain at least one tissuemicroarray, and at least one replicate of the tissue microarray.Replicates contained in such kits may be made, for instance, usingmethods described herein.

[0026] Also provided are kits for replicating a pattern of biomoleculesfrom a tissue sample, which kits include a plurality of membranes, eachhaving a coating on its upper and/or lower surfaces to increase specificbinding of a target biomolecule, a quantity of transfer buffer, and afluid impervious enclosure (for instance, a heat-sealable bag).

[0027] Another example of a described kit is a kit for analyzing aproteome, which kit contains a plurality of membranes, each having aaffinity for at least one protein, and a plurality of reagent species(such as detector molecules, particularly labeled detectors), eachspecies is adapted to detect one or more specific proteins bound to themembranes.

[0028] Further embodiments are membranes unit for use in blotting, whichunit includes a stack of at least two porous membranes (examples ofwhich have a thickness no greater than about 30 microns), and a frame,mounted to the membranes, which has a thickness no greater than about300 microns.

[0029] Also provided are porous membranes having a high affinity but lowcapacity for biomolecules. Examples of such membranes include a coresubstrate and a coating, and generally are no more than about 30 micronsthick. Specific examples of such membranes contain polycarbonate in thecore substrate and nitrocellulose in the coating.

[0030] The foregoing and other advantages and features will becomehereinafter apparent, and may be more clearly understood by reference tothe following detailed description, the appended claims, and the severalviews illustrated in the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031]FIG. 1 is a perspective view of a membrane array, showntransferring molecules from a tissue section using wicking transfer.

[0032]FIG. 2A is an oblique view of an apparatus shown transferringmolecules from a tissue section onto a membrane stack. FIG. 2B is afront view of an assembled contact transfer stack, prepared for transferin the apparatus illustrated in FIG. 2A.

[0033]FIG. 3 is a photograph of a typical tissue microarray on a slide.

[0034]FIG. 4A is a schematic illustration showing the components of akit according to one embodiment FIG. 4B is a perspective view of amembrane stack.

[0035]FIG. 5 is a schematic illustration showing a method according toone provided gel-transfer embodiment.

[0036]FIG. 6 is a sectional view of a stack of membranes shownoperatively engaged with an apparatus to transfer proteins from a gelonto the membranes.

[0037]FIG. 7A is a perspective view of a typical prior art LCMinstrument. FIG. 7B is an enlarged perspective view of an LCM cap shownengaged with a glass slide via a transport arm. FIGS. 7C and 7D are sideelevation views showing the transfer of cellular material from a tissuesection on a glass slide to an LCM cap.

[0038]FIG. 8 is a longitudinal section view of one embodiment, in whichLCM samples have been prepared for transfer through a membrane stack.

[0039]FIG. 9 is perspective view of one LCM transfer embodiment, shownin use and operation.

[0040]FIG. 10A is a side elevation view of a modified LCM cap accordingto provided embodiments. FIG. 10B is a section view taken along line B-Bof FIG. 4A.

[0041]FIG. 11 is a perspective view of a transfer array shown in usewith a microtiter plate.

[0042]FIG. 12 is a longitudinal sectional view of an individual membraneaccording to one provided embodiment.

[0043]FIG. 13 is a schematic drawing, illustrating direct capture.

[0044]FIG. 14 is a schematic drawing, illustrating indirect capture.

[0045]FIG. 15 is a schematic illustration showing a method according toanother gel-transfer embodiment

[0046]FIG. 16 is a perspective view of a representative framed membranestack.

[0047]FIG. 17 is a front elevation view of a single framed membrane.

[0048]FIG. 18 is a sectional view of the single membrane taken alongline 115-115 of FIG. 17.

[0049]FIG. 19 is a schematic illustration showing a hypothetical exampleillustrating the method of creating the antibody cocktails. The Gel (A)shows proteins as detected by Coomassie Blue staining prior to transfer.Membrane-Layer #1 (B), Membrane-Layer #2 (C), and Membrane-Layer #3 (C)show proteins detected on membranes with antibodies.

[0050]FIG. 20 is a schematic illustration showing the components of akit according to one embodiment.

[0051]FIG. 21 is an oblique view of a pressure heater apparatus.

[0052]FIG. 22 is a longitudinal section view of a stack of membranesshown with apparatus to transfer proteins from a gel onto the membranes.

[0053]FIG. 23 is a schematic illustration of one embodiment in use andoperation, showing the transfer of proteins from a gel to the membranestack so as to create multiple replicates of the protein content of thegel.

[0054]FIG. 24 is a sectional view of a stack of membranes shownoperatively engaged with an apparatus to transfer proteins from a gelonto the membranes.

[0055]FIG. 25 is a schematic illustration showing a comparison between atemplate image with a sample membrane.

[0056]FIG. 26 is a schematic illustration comparing the binding capacityof membranes constructed of nitrocellulose and polycarbonate, bothcoated and uncoated. FIG. 26A shows scanned images of the membranesincubated in protein comparing the intensity of signal. FIG. 26B is achart plotting the amount of protein bound to different membranematerials.

[0057]FIG. 27 shows images of tissue sections that show that portions oftotal biomolecules can be successfully transferred through a stack ofpolycarbonate (PC) layers onto the trap. FIG. 27A shows transfer throughpolycarbonate membranes. FIG. 27B shows transfer through polycarbonatecoated with nitrocellulose. FIG. 27C shows transfer throughpolycarbonate coated with poly-L-lysine membranes.

[0058]FIG. 28 is a series of images showing immunodetection of differentproteins from two regions (healthy and cancerous) of a breast tissueusing the membrane array.

[0059]FIG. 29 is a series of photographs of four membrane replicates ofa tissue microarray. The top row shows total protein staining of thereplicates with a ubiquitous stain; the bottom row shows immunodetectionof two specific proteins, keratin and prostate specific antigen (PSA).

[0060]FIG. 30 is a series of photographs showing a tissue microarraybefore transfer (stained with hematoxylin and eosin (H&E)) and fourreplicates thereof immunodetected with antibodies to four differentproteins (keratin, PSA, p53, and p300) as indicated.

[0061]FIG. 31 is a series of photographs showing total proteins capturedon the membranes (first column) and immunodetection of cytokeratin(second column).

[0062]FIG. 32 is a photograph of images of the membranes withbiotinylated protein bound to them. Proteins were separated by 1-D PAGE,transferred through the membrane stack and visualized withstreptavidin-alkaline phosphatase complex (strep-AP) and enhancedchemiluminescence (ECL) reagent.

[0063]FIG. 33 is a photograph of images of the membranes with Rsk andp300 proteins bound to them. Protein separation and blotting wasperformed as stated in FIG. 15.

[0064]FIG. 34 is a photograph of images of the membranes with GAPDH,Alpha-tubulin and Beta-actin bound to them. Proteins were separated by2-D PAGE, transferred through the membrane stack and visualized withprimary-secondary antibody-alkaline phosphatase complex and ECL reagent.

[0065]FIG. 35 is a photograph of images of the membranes with protein orDNA attached to them and a diagram that explains the relationshipbetween different protein-DNA complexes and their position in the gel.

[0066]FIG. 36 is line graph showing the relationship between proteinloading on the gel, protein size, and uniformity of transfer to themembranes.

[0067]FIG. 37 is a photograph showing differential expression ofgel-separated proteins from three cell samples (Jurkat, HN12, and SW480)blotted onto a seven-layer stack of membranes.

[0068]FIG. 38 is a photograph showing differential expression ofgel-separated proteins from four cell samples blotted onto a ten-layerstack of membranes. The upper row (marked “Total Staining”) shows themembranes stained ubiquitously with a dye. The bottom row (marked “ECL”)shows the membranes probed with the indicated antibodies.

[0069]FIG. 39 is a photograph showing distribution of total proteintransferred by a method provided herein.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0070] SEQ ID NO: 1 shows the nucleic acid sequence of a 43-residuesynthetic hybridization oligonucleotide.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0071] I. Explanation of Certain Terms

[0072] “Addressable” refers to that which is capable of being reliablyand consistently located and identified, as in an addressable locationon an array or a gel.

[0073] “Affinity” means the chemical attraction or force betweenmolecules.

[0074] “Antibody cocktails” means mixtures of between two to about 100different detector antibodies.

[0075] “Array” means two or more.

[0076] “Biological sample” means any solid or fluid sample obtainedfrom, excreted by or secreted by a living organism (includingmicroorganisms, plants, animals, and humans).

[0077] “Biomolecules” are molecules typically produced by livingorganisms including peptides, proteins, glycoproteins, nucleic acids,fatty acids, and carbohydrates.

[0078] “Capacity” means the ability to receive, hold, or absorbbiomolecules from the sample.

[0079] “Captor” means a molecule, such as an antibody or DNA probe, thatis anchored to a membrane and has an affinity (such as a specificaffinity) for one of the biomolecules.

[0080] biomolecule is not directly conjugated to the membrane.

[0081] “cDNA” refers to a DNA molecule lacking internal, non-codingsegments (introns) and regulatory sequences which determinetranscription. cDNA may be synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

[0082] “Counter-ligand staning” is intended to refer to any detectiontechnique that detects the presence of ligand that is not bound to aprotein of the biological sample, and thus reveals (as, for example, byan absence of staining, etc.) the presence of ligand that is bound to aprotein of the biological sample

[0083] “Detector” means a molecule, such as an antibody or DNA probe,that is free in solution (i.e. not anchored to a membrane) and has anaffinity for one of the sample components.

[0084] “Direct capture” means the conjugation or binding of abiomolecule directly onto the surface of the membrane without the aid ofa captor antibody or the like.

[0085] “DNA” is a long chain polymer that contains the genetic materialof most living organisms (the genes of some viruses are made ofribonucleic acid (RNA)). The repeating units in DNA polymers are fourdifferent nucleotides, each of which includes one of the four bases(adenine, guanine, cytosine and thymine) bound to a deoxyribose sugar towhich a phosphate group is attached. Triplets of nucleotides (referredto as codons) code for each amino acid in a polypeptide, or for a stopsignal. The term “codon” is also used for the corresponding (andcomplementary) sequences of three nucleotides in the mRNA into which theDNA sequence is transcribed.

[0086] “EST” (Expressed Sequence Tag) is a partial DNA or cDNA sequence,typically of between 500 and 2000 sequential nucleotides, obtained froma genomic or cDNA library, prepared from a selected cell, cell type,tissue or tissue type, organ or organism, which corresponds to an mRNAof a gene found in that library. An EST is generally a DNA moleculesequenced from and shorter than the cDNA from which it is obtained.

[0087] “Fluorophore” refers to a chemical compound, which when excitedby exposure to a particular wavelength of light, emits light (i.e.,fluoresces), for example at a different wavelength. Fluorophores can bedescribed in terms of their emission profile, or “color.” Greenfluorophores, for example Cy3, FITC, and Oregon Green, are characterizedby their emission at wavelengths generally in the range of 515-540 L Redfluorophores, for example Texas Red, Cy5 and tetramethylrhodamine, arecharacterized by their emission at wavelengths generally in the range of590-690 λ.

[0088] Examples of fluorophores that may be used are provided in U.S.Pat. No. 5,866,366 to Nazarenko et al., and include for instance:4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid, acridine andderivatives such as acridine and acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS), N-(4-anilno-1-naphthyl)maleimide, anthranilamide, BrilliantYellow, coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-trifluoromethylcouluarin (Coumaran 151); cyanosine;4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostibene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (AM),5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine;IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone;ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoetythrin; o-phthaldialdehyde; pyrene and derivatives such aspyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red4 (Cibacron .RTM. Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acidand terbium chelate derivatives.

[0089] Other suitable fluorophores include GFP (green fluorescentprotein), Lissamine™, diethylaminocoumarin, fluorescein chlorotriazinyl,naphthofluorescein, 4,7-dichlororhodamine and xanthene and derivativesthereof. Other fluorophores known to those skilled in the art may alsobe used.

[0090] “High throughput genomics” refers to application of genomic orgenetic data or analysis techniques that use microarrays or othergenomic technologies to rapidly identify large numbers of genes orproteins, or distinguish their structure, expression, or function fromnormal or abnormal cells or tissues.

[0091] “Hybridization” refers to an interaction between nucleic acidmolecules that are complementary to each other. Hybridization is basedon hydrogen bonding, which includes Watson-Crick, Hoogsteen or reversedHoogsteen hydrogen bonding between complementary nucleotide units. Forexample, adenine and thymine are complementary nucleobases that pairthrough formation of hydrogen bonds. “Complementary” refers to sequencecomplementarity between two nucleotide units. For example, if anucleotide unit at a certain position of an oligonucleotide is capableof hydrogen bonding with a nucleotide unit at the same position of a DNAor RNA molecule, then the oligonucleotides are complementary to eachother at that position. The oligonucleotide and the DNA or RNA arecomplementary to each other when a sufficient number of correspondingpositions in each molecule are occupied by nucleotide units which canhydrogen bond with each other.

[0092] “Specifically hybridizable” and “complementary” are terms thatindicate a sufficient degree of complementarity such that stable andspecific binding occurs between the oligonucleotide and the DNA or RNAtarget An oligonucleotide need not be 100% complementary to its targetDNA sequence to be specifically hybridizable.

[0093] Hybridization conditions resulting in particular degrees ofstringency will vary depending upon the nature of the hybridizationmethod of choice and the composition and length of the hybridizing DNAused. Generally, the temperature of hybridization and the ionic strength(especially the Na+ concentration) of the hybridization buffer willdetermine the stringency of hybridization. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are discussed by Sambrook et al. Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press (1989), chapters9 and 11, herein incorporated by reference

[0094] “Indirect capture” means the conjugation or binding of abiomolecule onto a captor antibody or the like which in turn is bound tothe surface of the membrane. Thus, with indirect capture the biomoleculeis not directly conjugated to the membrane.

[0095] “Identical” means having substantially the same affinity forbiomolecules.

[0096] “Label” refers to detectable markers or reporter molecules, whichcan be attached for instance to a specific biomolecule (e.g., a proteinor nucleic acid). Typical labels include fluorophores, radioactiveisotopes, ligands, chemiluminescent agents, metal sols and colloids, andenzymes. Methods for labeling and guidance in the choice of labelsuseful for various purposes are discussed, e.g., in Sambrook et al., inMolecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1989) and Ausubel et al., in Current Protocols in MolecularBiology, Greene Publishing Associates and Wiley-Intersciences (1987).

[0097] “Nucleic acid” refers to a deoxyribonucleotide or ribonucleotidepolymer in either single or double stranded form, and unless otherwiselimited, and encompasses known analogues of natural nucleotides thathybridize to nucleic acids in a manner similar to naturally occurringnucleotides.

[0098] “Membrane” means a thin sheet of natural or synthetic materialthat is porous or otherwise at least partially permeable tobiomolecules.

[0099] “Microarray” is an array that is miniaturized so as to requiremicroscopic examination for visual evaluation.

[0100] “Polypeptide” means any chain of amino acids, regardless oflength or post-translational modification (e.g., glycosylation orphosphorylation).

[0101] “Proteomics” means the identification or analysis of a proteome.A proteome is the group of proteins expressed and/or present in abiological sample.

[0102] “Sample” means a material that contains biomolecules includingtissue, gels, bodily fluids, and individual cells in suspensions or inpellet, as well as materials in containers of biomolecules such asmicrotiter plates.

[0103] “Stack” refers to adjacent substrates, whether orientedhorizontally, vertically, at an angle, or in some other direction. Thesubstrates (e.g., membranes) may be spaced or touching, for examplecontiguous.

[0104] “Subject” refers to living, multicellular vertebrate organisms, acategory that includes both human and veterinary subjects for example,mammals, birds, and more particularly primates.

[0105] II. General Description of Several Embodiments

[0106] Particular embodiments are especially useful in connection witharchival tissue samples that have been fixed and embedded, for instancein paraffin. Whole tissue sections, tissue macroarrays, and arrays ofminute tissue sections, e.g., in the format of a tissue microarray, allmay be analyzed according to the disclosed methods, as can other samplesfrom which biomolecules are to be detected (e.g., gels produced from 1-or 2-D separation of proteins or nucleic acids). The biomolecules on thecopies can be visualized using detector molecules (“probes”), forexample antibodies, lectins, or DNA hybridization probes, havingspecific affinity for the biomolecule(s) of interest.

[0107] Specific embodiments provided herein include direct layeredexpression scanning techniques, which utilize a stack of “blank”membranes that are not specific for any particular target molecule.Instead, all (or a subset, e.g., proteins or nucleic acid) biomoleculesin a sample ubiquitously bind to such membranes so as to give the userthe flexibility of detecting a wide variety of biomolecules in an openformat.

[0108] Thin membranes in a stacked or layered configuration are appliedto the sample, such as a tissue section, or protein or nucleic acid gel,and reagents and reaction conditions are provided so that at least aportion of the biomolecules are eluted from the sample and transferredonto a plurality of the stacked membranes. This produces multiplesubstantial replicas of the biomolecular content of the sample. Theresultant loaded (treated) membranes (or layers) are then separated.Each membrane may be incubated with one or more different detectors (forexample antibodies) specific for a particular biomolecule (such as aprotein) of interest. The detectors employed are labeled or otherwisedetectable using any of a variety of techniques, for instancechemiluminescence.

[0109] In an example in which proteins are detected, each membrane hasessentially the same pattern of proteins bound to it, but differentcombinations of proteins are made visible (detectable) on each membranedue to the particular detectors (e.g., antibodies) selected to beapplied. For example, one membrane layer may display proteins involvedin programmed cell death (apoptosis) while an adjacent layer may displayenzymes involved in cell division such as tyrosine kinases.

[0110] In addition to proteins, nucleic acids may be targeted by usinglabeled DNA probes as detectors in lieu of antibodies. Moreover,different types of target biomolecules may be detected in differentlayers. For example, both protein and nucleic acid targets can bedetected in parallel by applying protein-specific detectors (e.g.,antibodies) and nucleic acid detectors (e.g. hybridization probes) todifferent layers of the array.

[0111] According to certain methods of the present disclosure, a samplefrom which biological molecules are to be transferred (e.g., a tissuesection or gel) is positioned in contact with a face of a stack ofmembranes and both the sample and stack (an assembled “contact transferstack”) are placed inside a fluid impervious enclosure such as a plasticbag or the like. In certain embodiments, the sample is supported by asubstantially fluid impervious support, such as a glass slide; in theseembodiments, the stack of membranes is placed on the other side of thesample. In other embodiments, the sample from which biomolecules are tobe transferred is not supported by an impervious support, and the sampleis placed between members of the membrane stack, such that one or moremembranes is placed adjacent to each of two faces of the sample.

[0112] Also within the enclosure is a liquid transfer reagent. Heatand/or pressure are applied to the contents of the enclosure (from oneor both sides) so as to permit proteins and other molecules to betransferred from the sample to the membrane stack. This producesmultiple copies or replicas of the biomolecular content of the tissuesample. The processed membranes (or layers) then may be separated andincubated with one or more different probes (e.g. nucleic acidhybridization probes or antibodies) specific for particular targets ofinterest. The probes employed are labeled or otherwise detectable usingany of a variety of techniques such as chemiluminescence.

[0113] While each membrane has essentially the same pattern ofbiomolecules (including proteins and/or nucleic acids) bound to it,different combinations of such biomolecules are made visible on eachmembrane due to the particular probes or antibodies selected to beapplied. For example, one membrane layer may be used to detect proteinsinvolved in programmed cell death (apoptosis), while an adjacent layermay be used in detecting enzymes involved in cell division, such astyrosine kinases. In addition to proteins, nucleic acids may be targetedby using labeled DNA probes in lieu of antibodies. Moreover, bothprotein and nucleic acid targets can be detected in parallel by applyingboth antibodies and probes to different layers of the array of membranesto which the biomolecules have been transferred.

[0114] In one embodiment, the disclosed methods may be used for aside-by-side comparison of the protein expression patterns in differentarchival tissue samples, for instance from patients with differentdiseases, disease outcomes, or responses to therapies. Thus, forexample, where patient response to a particular drug can be correlatedto a specific protein expression pattern from the diseased organ thisprovides a useful tool for predicting whether future patients likelywill benefit or be harmed by that drug.

[0115] Advantageously, provided methods may be used to screen archivaltissue, which is usually formalin fixed and paraffin embedded. Providedmethods may also be used for examination of proteins that cannot bedetected with antibodies in situ but can be detected after the proteinhas been transferred onto a membrane. Furthermore, provided methodsenable the quantitative analysis of targets in tissue, for example, thequantification of cell surface receptor density on malignant cells.

[0116] Beneficially, the methods, device, arrays, and kits providedherein can be used with laser capture microdissected samples, permittingmolecular analysis of tissue without protein or nucleic acidpurification as a prerequisite. These embodiments retain thetwo-dimensional relationship of distinct cell populations within thesame tissue section so as to preserve the spatial relationships betweenthe dissected cells and permit different cell types to be processed andanalyzed in parallel.

[0117] Thus, methods are provided for detecting biomolecules in a samplecollected by LCM, by eluting the biomolecules away from themicrodissected sample and binding them to one or more membranes in alayered or stacked configuration, then visualizing the biomolecules onthe membranes.

[0118] In examples of such methods, cellular samples embedded in/on anLCM transfer film (or the like) are positioned adjacent to a stack ofone or more membranes, and reagents and reaction conditions are providedso that the biomolecules are eluted from the cellular sample andtransferred onto the membrane(s). Biomolecules on the membrane then canbe detected and visualized using detector molecules (e.g., antibodies orDNA probes) having specific affinity for the biomolecule(s) of interest.

[0119] Also provided are methods for identifying and analyzingbiomolecules that have been resolved via electrophoretic,chromatographic, or fractionating means. Examples of such methods aresensitive enough to detect proteins in low abundance, yet able to detectlarge numbers of proteins in a high-throughput manner preferably withoutrequiring expensive and sophisticated laboratory equipment.

[0120] Thus, according to one aspect of a method of the presentdisclosure, biomolecules (e.g., proteins or nucleic acids) that havebeen electrophoretically separated on a gel are transferred from the gelonto a stack of membranes. In certain examples, these membranes areconstructed and/or chemically treated to have a high affinity but lowcapacity for the biomolecules. This allows the creation multiplereplicates of the molecular content of the gel. After transfer, themembranes are separated and each is incubated with a one or a uniquemixture (also referred to as a “cocktail”) of detectors (e.g.,antibodies specific for a particular subset of proteins, nucleic acidprobes, etc). Thus, while each membrane has essentially the same patternof biomolecules bound to it, different combinations are made visible oneach membrane due to the particular detector (or set of detectors)selected to corresponds to the particular layer. In specific examples,the detector cocktail is an antibody cocktail that has been carefullyformulated so that no two antibodies in a cocktail bind overlapping oradjacent protein spots. Thus, protein spots that are too close togetherto be discriminated on a single membrane are detected on separatemembranes according to the inventive method herein.

[0121] According to certain disclosed methods, proteins that have beenseparated (either by in situ synthesis, electrophoretically,chromatographically, etc.) on a gel, tissue or other support aretransferred from the gel/support onto the membrane stack to allow thecreation of multiple replicates or imprints of the protein content ofthe gel/support. With regard to gels, the amount of protein loaded intothe wells is greater than the amount conventionally loaded so as topermit a more even and uniform distribution of the proteins throughoutthe stack.

[0122] Since antibodies can be used to detect many post-translationalprotein modification (e.g. phosphorylation), certain examples ofdisclosed methods can be employed to identify or analyze proteinfunction as well as structure. In addition to 2-D gels, describedmethods can be used for one-dimensional gels such as the identificationof transcription factors separated by a gel-shift assay.

[0123] In detail, one specific embodiment is a method of analyzing theproteome of a biological sample. Such a method involves separating theprotein from another protein present in the sample; transferring aportion of the separated protein to a plurality of membranes (forinstance, 2, 10, 20 or more) in a stacked configuration; incubating eachof the membranes in the presence of one or more species of predeterminedligand molecules (e.g., 2, 10, 20 or more) under conditions sufficientto permit binding between the separated protein and a ligand capable ofbinding to such protein; and analyzing the proteome by determining theoccurrence of binding between the protein and any of the species ofpredetermined ligand molecules.

[0124] Another embodiment is a method for analyzing the extent ofsimilarity between the proteomes of two or more samples. Such a methodinvolves, for each such sample, separating a protein of such sample fromanother protein present in the sample; transferring a portion of theseparated protein to a plurality of membranes (e.g., 2, 10, 20 or more)in a stacked configuration; incubating two or more of the membranes inthe presence of one or more species of predetermined ligand molecules(e.g., 2, 10, 20 or more) under conditions sufficient to permit bindingbetween the separated protein and a ligand capable of binding to suchprotein; and analyzing the extent of similarity between the proteomes bycomparing the separated proteins of each such sample with the separatedproteins of another such sample for the occurrence of binding betweenthe separated protein and any of the species of predetermined ligandmolecules.

[0125] Another embodiment is a method for uniquely visualizing a desiredpredetermined protein if present in a biological sample. This methodinvolves separating the proteins present in the sample from one another;transferring a portion of the separated proteins of the sample to aplurality of membranes (for instance, 2, 10, 20 or more) in a stackedconfiguration; incubating two or more of the membranes in the presenceof one or more species of predetermined detector/ligand molecules (e.g.,2, 10, 20 or more) under conditions sufficient to permit binding betweendesired predetermined protein and a ligand capable of binding to suchprotein; and visualizing any binding between the protein and any of thespecies of predetermined ligand molecules.

[0126] Also provided are embodiments of all such methods wherein theseparation of the protein from another protein present in the sample isaccomplished by electrophoresis (for instance, 2-dimensional (2-D) gelelectrophoresis).

[0127] Further embodiments include all such methods wherein the sampleis obtained from mammalian cells or tissue, and particularly from humancells or tissue, and the embodiments wherein the mammalian cells ortissue are human cells or tissue and the separated protein is a productof a human gene.

[0128] It is contemplated that the detector/ligand species can be any ofa variety of molecule types. Thus, also provided are embodiments of allsuch methods wherein at least one of the species of detector/ligand isan antibody, an antibody fragment, a single chain antibody, a receptorprotein, a solubilized receptor derivative, a receptor ligands, a metalion, a virus, a viral protein, an enzyme substrate, a toxin, a toxincandidate, a pharmacological agent, a pharmacological agent candidate, ahybridization probe, a oligonucleotide, and others as discussed herein.

[0129] Other embodiments include all such methods wherein the binding ofat least one of the species of detector/ligand is dependent upon thestructure of the separated biomolecule (e.g., protein or nucleic acid).It still further provides the embodiments of all such methods whereinthe binding of at least one of the species of detector/ligand isdependent or upon the function of the separated biomolecule (e.g.,protein or nucleic acid).

[0130] The disclosure also provides all such methods wherein at leastone of the membranes is incubated with more than one species of ligandor detector molecule. Also provided are embodiments of all such methodswherein at least two membranes are employed, at least 10 membranes areemployed, or at least 20 membranes are employed.

[0131] Further provided are the embodiments of all such methods whereinat least at least two ligand species or detector molecules are employed,wherein at least 10 are employed, or at least 20 or more are employed.

[0132] Additional embodiments are membranes that have a high affinitybut a low capacity for proteins and/or other biomolecules so as to allowthe creation of multiple replicates or imprints of the proteins elutedfrom a gel. Examples of these membranes are substantially thinner thanthose conventionally used for blotting. The membranes are optionallyprovided with (or within) a frame, so that they may be easily handledand manipulated when separated from that stack. The frame optionallydefines a channel to permit release of air and fluid trapped betweenadjacent membranes. Removable tabs or the like also may be provided oneach frame to permit the stack to be held together, for instance when itis applied to the gel.

[0133] Loaded membranes may be scanned or otherwise digitally imagedusing one of several commercially available scientific imaginginstruments. Imaging instrumentation and software, such as thosedescribed herein, may be employed to permit viewing, analysis, and/orinterpretation of the expression patterns from the sample (e.g., atissue sample or other two-dimensional source, such as a gel). Softwaremay be provided with template images corresponding to each of themembrane images. This allows the identity of the biomolecule in eachdefined locus (e.g., a spot on a 2-D gel, a band on a 1-D gel, or alocalized molecular deposit in a tissue sample) to be confirmed based onits vertical and horizontal position. The software also can allow thedensity of each locus to be calculated so as to provide a quantitativeread-out The software may also have links to a database of imagesgenerated from other gels to allow comparisons to be made betweendifferent diseased and normal samples. In addition to computerizedanalysis of membranes, the source sample (e.g., actual tissue sectionsor other substantially two-dimensional source) or a substantiallysimilar sample (e.g., an adjacent tissue slice) may be analyzed withconventional techniques (e g., histochemical techniques) to confirm orcompare the digital analysis.

[0134] Also provided herein are kits that include a plurality ofmembranes (e.g. 3 or more, for instance 5, 10, 15, 25, 50, or 100 ormore membranes) in a stack or other configuration that permits them tobe stacked. Optionally, the provided kits may further include one ormore different detectors, such as cocktails of antibodies orhybridization probes, to be applied to the treated membranes forbiomolecule detection/analysis. The kits may also provide one or moreadditional components, such as a volume of a transfer reagent, a fluidimpervious enclosure (for instance, a sealable bag), one or more piecesof filter paper, and/or a tissue array contained on a slide or othercomparison sample or control sample. Optionally such kits may alsoinclude instructions for how to use the kit to detect, analyze, and/oridentify one or more biomolecules. Detection chemistries may beincluded, which are tailored to coincide with the detector moleculesprovided with the kit or anticipated for use with the other kitcomponents. The aforementioned software may also be included in the kit,or may be accessible via modem or the Internet.

[0135] In certain embodiments, the methods and kits according to thepresent disclosure allow up to several thousand discrete biomolecule(e.g., protein) loci to be identified, annotated, and, at the user'soption, compared to the pattern of loci generated from other samplesstored in a database.

[0136] One specific example of a provided kit for analyzing a proteomeincludes a plurality of membranes, each having a specific affinity forat least one protein, and a plurality of detector/ligand species (e.g.,species such as an antibody, an antibody fragment, a single chainantibody, a receptor protein, a solubilized receptor derivative, areceptor ligands, a metal ion, a virus, a viral protein, an enzymesubstrate, a pharmacological agent, and a pharmacological agentcandidate), each adapted to detect one or more specific proteins boundto the membranes.

[0137] Also provided in another embodiment is a kit for uniquelyvisualizing a desired predetermined protein if present in a biologicalsample. Such a kit includes a plurality of membranes, each having aspecific affinity for at least one protein, and a plurality ofdetector/ligand species (e.g., species such as an antibody, an antibodyfragment, a single chain antibody, a receptor protein, a solubilizedreceptor derivative, a receptor ligands, a metal ion, a virus, a viralprotein, an enzyme substrate, a pharmacological agent, and apharmacological agent candidate), each adapted to detect the desiredpredetermined protein if bound to the membranes.

[0138] In particular embodiments, the membranes provided in kitsdescribed herein include a porous substrate having a thickness of lessthan about 30 microns. Particular examples of such a kit includemembranes that are polycarbonate membranes, especially polycarbonatemembranes coated with a material for increasing the affinity of themembrane to biomolecules, for instance nitrocellulose, poly-L-lysine, ormixtures thereof.

[0139] III. Transfer Modes

[0140] Provided herein are multiple methods for transferringbiomolecules from a sample that is generally substantiallytwo-dimensional into one or more thin membranes, usually arranged in astack. Several different specific transfer modes are provided. Some ofthese modes overlap, in that wicking or contact transfer can be used totransfer biomolecules from both tissue- and gel-based samples, and soforth. Even though perhaps not explicitly enumerated, all variations andcombinations of the described methods are encompassed herein.

[0141] Wicking Transfer

[0142] In particular embodiments, a transfer liquid (such as a buffer)is passed through the membranes to encourage movement of thebiomolecules from the sample to the membranes and through them. A distalor downstream wick may also be provided to help move liquid (such as thebuffer) through the membranes in a desired direction of movement

[0143] There is illustrated in FIG. 1 a perspective view of arepresentative disclosed membrane array transfer apparatus designatedgenerally by reference numeral 10. Apparatus 10 includes a plurality ofmembranes 12 shown in a layered or stacked configuration such as array13. While only about a dozen membranes are shown in array 13 of FIG. 1,it will be appreciated that many more membranes (e.g., 10, 50, 100 ormore) may be employed depending on the number of targets sought to beidentified, the quantity of biomolecules present in the sample, and thethickness of the material employed to construct membranes 12.Optionally, membranes 12 may be packaged in a suitable sealed enclosureor frame (not shown), for instance to maintain their integrity and/orprevent contamination.

[0144] Membrane array 13 is placed atop a stack of one or more sheets ofblotting paper 14 that acts as a lower wick pulling buffer out of bufferchambers 18 though upper wicks 20 and membrane array 12 in the directionof the arrows shown in FIG. 1. A biomolecule trap 22 may be positionedintermediate membrane array 12 and blotting paper 14 to help the userascertain whether and/or to what extent transfer has occurred.

[0145] In use and operation, apparatus 10 may be employed to create“carbon copies” or substantial replicas of the biomolecular contents ofthe sample applied to the stack. Membranes 12 are arrayed in a layeredor stacked configuration as shown in FIG. 1 as reference numeral 13. Ina particular embodiment, a substantially two-dimensional sample 11 (suchas a conventional frozen tissue section as illustrated) is placed on asupport substrate (e.g., a layer of polycarbonate) and then sandwichedbetween two slices of 2% agarose (not shown). The entire preparation ispositioned adjacent to the membrane array 13. Buffer 16 is applied usingbuffer chambers 18 and upper wicks 20 to elute and transfer proteinsfrom the frozen section. About 50-100 milliliters of buffer per squarecentimeter are used in each transfer with average length of the transferbeing about 1-2 hours.

[0146] After transfer the membranes are separated and incubated with thedetector antibody. Antibodies are selected based on the types of targetssought. Membranes are washed in a buffer and the protein/detectorcomplex can be visualized using a number of techniques such as ECL,direct fluorescence, or colorimetric reactions. Commercially availableflatbed scanners and digital imaging software can be employed to displaythe images according to the preference of the user.

[0147] The specific example illustrated in FIG. 1 shows a device and amethod for detecting biomolecules in a tissue section 11 or othertwo-dimensional sample (e.g. an electrophoretic gel) by creating “carboncopies” (substantial copies that are not necessarily identical copies,they may have slight differences but can be identical or nearlyidentical) of the biomolecules eluted from the sample, and visualizingthe biomolecules on the copies using antibodies or other moleculeshaving specific affinity for the biomolecules of interest. Thinmembranes 12 in a stacked or layered configuration are brought intocontact with the sample and reagents, and reaction conditions areprovided so that the biomolecules are eluted from the sample onto themembranes, whereupon the biomolecules can be visualized using a varietyof techniques, as set forth herein.

[0148] Certain embodiments of the disclosure include a method ofdetecting an analyte in a biological sample using stacked contiguouslayered membranes that permit biomolecules to move through a pluralityof the membranes, while directly capturing the biomolecules on one ormore of the membranes. Biomolecules from the sample are moved throughthe membranes under conditions that allow one or more of the membranesto directly capture the biomolecules, and biomolecules of interest areconcurrently or subsequently detected on the membranes, for example byexposing the biomolecules of interest to a detector, such as a specificcapture molecule (for example an antibody or a nucleic acid probe).

[0149] Alternatively, the biomolecule itself may be a detector (such asa nucleic acid probe) to which a sample is exposed. In this case, thebiological sample is one or more purified nucleic acid probes placed inassigned locations on a surface of the stack, which are allowed tomigrate through membranes (for example in a direction of movementtransverse to the layers) to produce multiple substantial “copies” ofthe original probes in corresponding locations on the multiplemembranes. The layers then can be separated and exposed to a targetbiological specimen, which may have nucleic acid molecules thathybridize to the probes.

[0150] In some examples, the biological sample is a tissue specimen thatis placed on the stack of layered membranes, and biomolecules from thetissue specimen are directly captured by the membranes as thebiomolecules move through the membranes. The membranes may, for example,be separated prior to detecting the biomolecules of interest, and theseparated membranes are exposed to the detectors. Alternatively, thebiological molecules of interest may be contained in a biologicalspecimen to which the membranes are exposed. For example, thebiomolecules directly captured by the membranes may themselves benucleic acid probes or antibodies, and the membranes may be exposed to abiological specimen in which a nucleic acid or peptide (such as aprotein) is to be detected.

[0151] Biomolecules detected on the membrane copies may be correlatedwith a biological characteristic of the sample. For example, a tissuespecimen may be placed in a position on top of the stack, and abiomolecule of interest (such as a particular protein) may be detectedin one of the membrane copies at a position that corresponds to theposition in which the tissue specimen (or one of its substructures suchas an organelle) was placed. The presence of that biomolecule in thetissue specimen can then be correlated with a biological characteristicof the sample. For example, a highly malignant tissue specimen may befound to contain a protein that may then be associated with the highlymalignant phenotype of the specimen.

[0152] In particular examples, the method can be used to create a set ofmicroarray substantial “copies” by applying a plurality of detectors,such as DNA probes, antibodies, or a combination thereof, to the stackof layered membranes. The stack of layered membranes provide a pluralityof substrates through which the probes or antibodies (generally,detector molecules) move, and in which a portion of the probes orantibodies are directly captured by one or more of the substrates. Thesubstrates can be subsequently separated to provide correspondingsubstrates having a plurality of DNA probes, antibodies or a combinationthereof in corresponding positions of each of said substrates. Themultiple membranes maintain a substantially coherent relationshipbetween the probes and/or antibodies as they move through the substrate.This coherent relationship may or may not be a direct spatialcorrespondence, but the relative relationship between the biomoleculesmay be maintained in such a way that the identity of the biomolecules onthe membranes can be known from the relationship in which thebiomolecules were placed on the stack of layered membranes.

[0153] Contract Transfer

[0154] There is illustrated in FIG. 2A an alternative embodiment of anapparatus 10 for transferring biomolecules from a substantiallytwo-dimensional sample 11 onto a membrane stack 13, which stack in someembodiments is provided in the form of a kit Apparatus 10 generallyincludes a membrane stack 13 upon which a sample 11 (illustrated as atissue section) may be placed, a pair of filter pads 24 and 26, and afluid impervious enclosure 28, such as a plastic bag or the like.Optionally, the sample 11 (e.g., a tissue section) may be presented on asupport 30 (as illustrated in FIG. 2B). In particular embodiments, thesupport 30 is a microscope slide or other fluid impervious support suchas a piece of tape.

[0155] More specifically, in a first embodiment, membrane stack 13comprises one or more membranes 12, for instance up to five membranes,generally constructed as described herein. The membranes 12 in stack 13should have a high affinity for proteins and other biomolecules but havea low capacity for retaining such molecules. This feature permits themolecules to pass through the membrane stack with only a limited numberbeing trapped on each of the successive layers, thereby allowingmultiple “carbon copies” (substantial copies that are not necessarilyidentical copies, they may have slight differences but can be identicalor nearly identical) to be generated. In other words, the low capacityallows the creation of multiple replicates as only a limited quantity ofthe biomolecules are trapped on each layer.

[0156] First and second filter pads 24, 26 are preferably constructed ofa blotting paper such as GB004 Blotter Paper available from Schleicherand Schuell. Filter pads 24, 26 are saturated with a transfer buffersuch as Tris or phosphate base buffers.

[0157] Enclosure 28 may comprise any collapsible, fluid imperviousmaterial adapted to envelop the sample 11, membrane stack 13, and filterpads 24, 26, which may be kit components. Enclosure 28 is preferably aplastic bag, such as a heat sealable pouch. By way of example, such abag may be made of a resin, such as a polyester or other resin. Incertain embodiments, enclosure 28 is a heat sealable pouch such as thoseavailable from Kapak Corp. (Minneapolis, Minn.).

[0158] In use and operation, the sample 11 (e.g., a tissue sectionsample or tissue microarray 31, shown in FIG. 3) is positioned incontact with a face of a membrane stack 13 and both the sample and stackare placed between two filter pads 24, 26, which have been saturatedwith transfer buffer, to for an assembled contact transfer stack. Theassembled contact transfer stack is placed inside fluid imperviousenclosure 28, such as a plastic bag. The membranes are pre-wetted in theaforementioned transfer solution.

[0159] Fluid impervious enclosure 28 is placed between a pair ofsubstantially flat surfaces 32, at least one of which also serves as asource of heat. By way of example, the pair of substantially flatsurfaces 32 can be surfaces of a pair of heating elements such as thoseprovided in gel dryers manufactured by Bio-Rad Laboratories (Hercules,Calif.). In other embodiments, the pair of flat surfaces 32 may beprovided by MI Research devices, such as the PTC-200 Peltier thermalcycler, which provide a separate heated lid and a thumbwheel to adjustheight and pressure of the lid and thereby provide pressure.

[0160] In embodiments where heat is applied only from one side of theassembled sample and stack, the heat is preferentially applied from theside of the sample rather than the membrane stack side, such that a heatgradient is created with the heat applied on the sample side.

[0161] To effect transfer, the bag and its contents are heated to atemperature of 60 to 95° C., in some embodiments 60 to 80° C., or moreparticularly in some embodiments 70° C. The bag and its contents areheated for at least about an hour, and in some embodiments about twohours or more. Sufficient pressure is applied throughout the heatingprocess to ensure that there is adequate contact between the sample andthe membrane stack to facilitate transfer of biomolecules to themembrane stack. By way of example, such pressure can be applied using aweight 34 of 0.5 to 2 pounds, which may optionally be included as a kitcomponent. Springs, clamps, or clips capable of applying pressure may beemployed instead of a weight.

[0162] The combination of heat and pressure being applied causesbiological components, including proteins and/or nucleic acids and/orcarbohydrates and/or lipids, to be transferred from the sample 11 tomembrane stack 13. This produces multiple copies or replicas of thebiomolecular content of the tissue sample, due at least in part to thebinding characteristics of the membranes.

[0163] To ensure that the binding capacity of the membranes issufficiently low to prevent trapping of too much of the sample, in someembodiments the thickness of membrane substrate should be less than 30microns, in some embodiments from 4 to 20 microns, and particularembodiments from 8 to 10 microns. The pore size of the substrate shouldbe from 0.1 to 5.0 microns, in particular embodiments 0.4 microns.Another advantage of using such a thin membrane is that is lessens thephenomenon of lateral diffusion. The thicker the stack of membranes, thewider the diffusion of biomolecules moving through the stack.

[0164] The substrate includes a coating on its upper and/or lowersurfaces to increase specific binding of the proteins or other targetedbiomolecules. The coating in certain embodiments is nitrocellulose, butother materials such as poly-L-lysine may also be employed.

[0165] Tissue section sample 11 may be derived from fresh/frozen tissueor tissue that has been fixed in formalin (or another fixative) andparaffin embedded tissue. The section is created by conventionalmethods, for instance using a microtome. The thickness of a tissuesection can vary from 3 to 30 microns depending on the desired number ofmembrane replicates to be created. As a rule of thumb, the thickness ofthe section should be one micron for each replicate sought. Thus, forexample, a 10 micron section would be used to create ten membranecopies.

[0166] As used herein “tissue” means any material containing cells,proteins, or nucleic acids including plant, animal, and human material.In lieu of tissue section sample 11, a tissue microarray 31 (FIG. 3) maybe employed. Tissue microarrays are described in Kononen et al, NatureMedicine, 4:844-847, 1998) and are provided by several commercialentities, such as the Vast Array™ tissue arrays available from ResearchGenetics (Huntsville, Ala.). Tissue macroarrays are similarlyconstructed, except that they contain tissue sections that are generallylarger than microarray samples; the tissue samples used in tissuemacroarrays may optionally be dissected by hand. Alternately, in someembodiments the biomolecules on a gel (e.g., an electrophoretic gel) orother substantially two-dimensional sample are transferred to a membranestack using similar methods, in place of tissue section 14.

[0167] Gel-Based Transfer

[0168] The most widely used method for identifying and measuringbiological molecules is gel electrophoresis, a collection of techniquesfor separating or resolving molecules in a mixture under the influenceof an applied electric field based on (usually) the difference in theirsize and/or charge. Electrophoretic separation is most commonlyperformed using porous polymer gels. During one-dimensionalelectrophoresis, a mixture of proteins is applied to a gel and exposedto the flow of an electric current. Since smaller proteins migratefaster through the gel than larger ones, separation based on their sizeis achieved. By way of example, this one-dimensional approach can onlygenerate about 100 distinct protein bands, which is inadequate for manyapplications since the estimated number of proteins expressed in atypical mammalian cell is between about 10,000-15,000 proteins.

[0169] In order to improve the resolving power of electrophoresis gels,a two-dimensional gel technique was introduced in the 1970s, whereinelectrophoretic separation of the proteins based on their size ispreceded by charge-based separation. Isoelectric focusing (EF)electrophoresis, which separates proteins according to their charge(pH), is run in one direction and mass separation is carried out in aperpendicular direction. Such two-dimensional (2-D) gel electrophoresis(often abbreviated as “2-D PAGE,” for two dimensional polyacrylamide gelelectrophoresis) has become the backbone of proteomics. The technique isroutinely employed for characterizing the proteome of different classesof tissues, cells, cell lysates, body fluids or exudates. The end resultof 2-D PAGE is the production and separation of various protein “spots”in a two dimensional Cartesian plane where the coordinates of each spotare represented by charge and molecular weight. However, the majorchallenge of 2-D electrophoresis is the identification of the proteinsafter they have been separated on the gel.

[0170] Proteins that have been separated on gels are usually identified,detected, and analyzed by one of several different techniques. If theprotein spot represents an unknown protein, the most common approach isto physically remove or excise the spot from the gel, digest it with anenzyme, and characterize the protein by mass spectroscopy. A computergenerates a plot of protein fragments according to their mass, and thisplot serves as a fingerprint that may be used to facilitate theidentification of the original protein. As in the analysis of actualfingerprints, the ability of mass spectroscopy to identify a detectedprotein relies on the prior recovery and analysis of a reference proteinwhose fragments match those of the detected protein. The identificationof a truly new protein by mass spectroscopy remains a significantchallenge.

[0171] Although mass spectroscopy provides the most incontrovertibledata, the method is time consuming, expensive and cannot be accomplishedin the absence of expensive core facilities and highly trainedpersonnel. Furthermore, the technique is used only to analyze theproteins that can be stained with a ubiquitous stain such as Coomassieblue. Unfortunately, ubiquitous stains are not sensitive and permit onlya small fraction of the proteins in the sample to be visualized. Inother words, mass spectroscopy of ubiquitously stained gels does notyield a broad “dynamic range” as it fails to identify certain lowabundance—but potentially important—proteins. Among the low abundanceproteins that may be left behind by these techniques are tyrosinekinases, cytokines, and transcription factors, which play a key role inmany diseases.

[0172] An alternative approach to identifying gel separated proteins isimmuno-blot analysis, which uses a detectable antibody specific to aprotein of interest in lieu of a ubiquitous stain. The proteins aretransferred onto a membrane, typically constructed of eithernitrocellulose or of polyvinylidene difluoride (PVDF) and antibodies areapplied to the membranes. Immuno-blotting is rapid and can beaccomplished in less than a day. Also, it is estimated to be about1000-fold more sensitive than Coomassie blue staining, allowing even lowabundance proteins to be identified. It is significantly more specificas well. However, a key limitation of immuno-blotting is that at mostonly a handful of proteins can be identified on a single blot due tooverlapping spots and cross-reactivity with different proteins in thesample. Since the 2-D gel process requires approximately 24 hours tocomplete, it would be prohibitively time consuming to create enoughimmuno-blots to identify the large quantity of proteins needed for mostproteomics applications.

[0173] Thus, there is a clear need to develop techniques that permitlarge numbers of proteins across a wide dynamic range to be identifiedin parallel. Information potentially relevant to attempts to addressthis need can be found in the following references: Sanchez et at,Electrophoresis, 18:638-641, 1997; Neumann & Mullner, Electrophoresis,19:752-757, 1998; Manabe et al., Annal. Biochem., 143:3945, 1984;Legocki & Verma, Annal. Biochem., 111:385-345, 1981; and PCTInternational Publication No. WOOO 045168, all herein incorporated byreference.

[0174] However, each of the techniques described in these referencessuffers from one or more of the following disadvantages: (i) notsensitive enough to detect low abundance proteins, (ii) cannot identifylarge numbers of proteins in a high-throughput manner, and (iii)requires specialized or sophisticated hardware that leads to loss ofprotein and a decrease in the resolution the protein spots during thetransfer.

[0175] According to methods provided herein, biomolecules that have beenelectrophoretically separated on a gel, or via chromatography, etc. aretransferred from the gel onto a stack of membranes. Examples of suchmembranes are membranes that are constructed and chemically treated tohave a high affinity but low capacity for proteins. Suitable membranesand methods for their construction and preparation are described herein.The use of such membranes allows the creation of multiple replicates ofthe protein content of the gel.

[0176] The membranes are then incubated with a unique ligand species (adetector molecule) or mixture or cocktail of such, to assist in andpermit detection and/or analysis of biomolecules on the membranes. Themembranes are generally separated one from another prior to suchincubation. Detector molecules/ligands can be any of a number ofmolecules that have binding specificity for a target molecule ofinterest, and include antibodies (such as monoclonal antibodies),antibody fragments (e.g., FAB, F(AB)₂, single chain antibodies, receptorproteins, solubilized receptor derivatives, receptor ligands, metal ions(particularly paramagnetic or radioactive ions), viruses, viral proteins(e.g., human rhinovirus or proteins thereof that bind to ICAM-1, or HIVor proteins thereof that bind to CD44), enzyme substrates, toxins, toxincandidates, pharmacological agents, pharmacological agent candidates,other small molecules that bind to specific proteins, as well asmolecules that bind or hybridize to nucleic acids (e.g., nucleic acidprobes or specific binding proteins or fragments thereof) etc. Whileeach membrane has essentially the same pattern of biomolecules bound toit, different combinations of such biomolecules can be detected on eachmembrane due to the particular ligand or cocktail of ligands selected tocorresponds to the particular layer.

[0177] The nature of the species of ligand(s) in the cocktail providedto the membrane determines the nature of information that can beobtained from that membrane. For example, by incubating a membrane withan antibody or antibody fragment, one is able to identify the presenceor absence of protein molecules of the sample that bind to suchmolecules. In this way, for example, a membrane could be incubated withan antibody that specifically binds a protein kinase, in order todetermine whether a particular protein is a protein kinase, or possessesan epitope that mimics that of a protein kinase. Similarly, by employingas the ligand, a cellular receptor protein, solubilized receptorderivative, or receptor ligand, the membrane would enable one toidentify whether a particular protein was a receptor or receptor ligand.Since viruses and other pathogens are capable of binding to cellularreceptor proteins, a cocktail containing a virus or viral protein couldbe employed in the same manner as a receptor ligand to identify whethera particular protein was a cellular receptor or receptor ligand. In analternative embodiment, the cocktail could comprise one or morepharmacological agents to identify proteins that interact with suchagents. Likewise, pharmacological agent candidates could be incubatedwith the membranes, thereby revealing the ability of such candidatemolecules to bind to specific proteins. For example, anacetylcholinesterase inhibitor or a monoamine oxidase inhibitor (MAOI)could be incubated with a membrane to identify proteins that bind theinhibitor and which thus might be additional therapeutic targets of theinhibitor. Likewise, a compound suspected of possessing therapeuticpotential could be incubated with a membrane to reveal whether it bindsto proteins expressed, for example, in the liver or kidney, therebyrevealing its potential to treat diseases affecting these organs.Examples of the methods and kits permit the further analysis of suchbinding to determine, for example, whether such proteins are expressedin other organs and tissues (e.g., the brain).

[0178] In one embodiment, a membrane will be incubated in the presenceof a single ligand, or a cocktail of different ligands of the same classof ligands (e.g., antibodies, receptors, hybridizing probes, etc.).Alternatively, a membrane may be incubated with different classes ofligands. For example, a membrane that is incubated with antibodies thatbind protein kinases and with a therapeutic candidate, can be employedto reveal therapeutic candidates that bind to protein kinases. Wheremixtures or cocktails of ligands are employed, the cocktails arepreferably formulated so that no two ligands bind overlapping oradjacent protein spots. Thus, for example protein spots that are tooclose together to be discriminated on a single membrane may be detectedon separate membranes.

[0179] In an alternative embodiment, the ligand is permitted to bind toproteins of the sample prior to the transfer to a membrane. Thus, insome examples the ligand is provided to a living or deceased subject, toa tissue or cell, to a tissue or cell preparation, or to a tissue orcell extract, prior to the fractionation or separation of protein. Theproteins are then transferred to membranes and the proteins and ligandare visualized. In such an embodiment, one can detect whether bindingbetween a ligand and a protein of the sample and occurs in situ, and/orunder physiological conditions. Optionally, the membranes can beincubated in the presence of additional ligand (which may be the same ordifferent from the initially employed ligand) in order to detectcompetition between or among ligands for binding sites, to evaluate theavidity of binding, to examine binding complexes of three or moremolecules, etc.

[0180] Particular embodiments provide a method and a kit 36 foridentifying (i.e. detecting, annotating, and/or characterizing) groupsof proteins (not shown) that have been separated by gel electrophoresis.As illustrated in FIG. 4A, in one example kit 36 generally comprises thefollowing components: (i) a stack of membranes 13 upon which theproteins are transferred, (ii) primary antibody cocktails 38, forinstance one for each of the membranes 13, and (iii) other reagents 40including (as in illustrated in kit 36) protein transfer buffer 42 andantibody detection chemistries 44. The kit 36 may also include software46 that allows the user to analyze and manipulate the images produced soas to yield a “proteomic image” of the biological sample being testedand compare it to proteomic images from other samples in a database.Alternatively the software may be acquired or accessed independent ofthe kit

[0181] In a specific embodiment, and with reference to FIG. 4A, membranestack 12 comprises a plurality of membranes 13 adapted to be removablystacked atop one another, as shown.

[0182] According to the method of a particular embodiment (asillustrated in FIG. 5), proteins 48 that have been electrophoreticallyseparated on gel 50 are transferred from the gel through membrane stack13. This allows the creation of multiple replicate blots 52 of theprotein content of the gel. The membranes are then separated and each isincubated with one of the unique cocktails 38(a-c) of ligands, e.g.,antibodies. The antibodies employed are labeled or otherwise detectableusing any of a several techniques such as enhanced chemiluminescence(ECL). This produces unique spot patterns 54(a-c) on each of themembranes. The membranes with unique spot patterns 54 are then scannedor digitally imaged using an imaging instrument (not shown) so that thedensity of the spot may be calculated, compared to other samples, anddisplayed on a computer using software 46, as described herein.

[0183] One advantage of specific embodiments provided herein is thatthey provide a third dimension of protein separation for a biologicalsample, one additional dimension from the size and charge separationsobtainable from 2-D gels. The layered membranes provide a cost-effectivetool for selecting groups of compatible antibodies that can be used todetect subsets of proteins on the same membrane. Once selected theseligand combinations can be packaged in a kit and used repeatedly for thecontrolled analysis of proteomes displayed on stacked membranes. Since15-20 replicates or copies can be generated from a single gel and ten ormore ligands can be applied to each membrane several thousand differentproteins can be identified from a single gel according herein describedmethods.

[0184] Since ligands can be used to detect many post-translationalmodification of proteins (e.g. phosphorylation) the present disclosurecan be employed to identify protein function as well as structure.

[0185] Although these embodiments have been described with respect to2-D gels, it is also contemplated that the methods and devices describedcan be employed with one dimensional gels (e.g., as for theidentification of transcription factors separated by a gel-shift assay),or proteins may be separated from other proteins of a sample, by othermeans, as by chromatography. It is also contemplated that these methodscan be used to generate duplicate copies of non-protein biomolecules,such as nucleic acids, lipids, sugars (such as polysaccharides) andcombinations or complexes of two or more types of biomolecules.

[0186] In certain embodiments, buffer reagent for eluting proteins froma gel to a membrane stack comprises a mixture of glycine, methanol, andSDS as described herein. For 1-D gel analysis, protein staining can becarried out using FastBlue Stain (Chemicon). Bi-Directional Transfer

[0187] In alternative embodiments of the provided methods, the samplefrom which biomolecules are to be transferred is not supported by animpervious support and the sample is placed between members of themembrane stack. Thus, in such embodiments one or more membranes isplaced adjacent to each of two faces of the substantiallytwo-dimensional sample, and transfer of the biomolecules from the sampleto the membranes occurs in two directions (bi-directional transfer).

[0188] By way of example, this technique is illustrated schematically inFIG. 6. Here first and second membrane stacks 13 a and 13 b sandwich gelslab 54, which contains sample 11. A pair of filter pads 24 and 26,preferably constructed of a blotting paper such as GB004 Blotter Paperavailable from Schleicher and Schuell are provided adjacent to themembrane stacks as shown. Filter pads 24 and 26 are saturated with atransfer buffer such as TLIS or phosphate base buffers.

[0189] A collapsible, fluid impervious enclosure 28 is provided toenvelop the pads, membrane stacks, and gel as shown in FIG. 7. Enclosure28 (which in some instances is a plastic bag) is preferably a heatsealable pouch/bag such as those available from Kapak Corp.(Minneapolis, Minn.). Preferably, most of the air is removed fromenclosure 28 by gentle squeezing and/or vacuum suction and it is sealedby a heat sealer such as the Impulse Sealer (American InternationalElectric). Enclosure 28 is then placed between a pair of heatingelements 56 a and 56 b such as those provided in Gel Dryers manufacturedby Bio-Rad Laboratories (Hercules, Calif.). The enclosure 28 and itscontents are optionally heated to a temperature of between about 50 to90° C., preferably to about 80° C. for about 2-4 hours. In someembodiments, pressure is applied throughout the heating process using aweight 34.

[0190] The heat and pressure applied to contents of the enclosure permitproteins and other molecules to be transferred from the gel or othertwo-dimensional sample to the membrane stack. This produces multiplecopies or replicas of the biomolecular content of the sample.

[0191] Transfer from Laser Capture Microdissection Samples

[0192] Under the microscope, tissues are heterogeneous, complicatedstructures with hundreds of different cell types locked in morphologicunits exhibiting strong adhesive interactions with adjacent cells,connective stroma, blood vessels, glandular and muscle components,adipose cells, and inflammatory or immune cells. In normal or developingorgans, specific cells express different genes and undergo complexmolecular changes both in response to internal control signals, signalsfrom adjacent cells, and humoral stimuli. In diseased tissues the cellsof interest, such as pre-cancerous cells or invading groups of cancercells, are typically surrounded by these heterogeneous tissue elements.Cell types undergoing similar molecular changes, such as those thoughtto be most definitive of the disease progression, may constitute lessthan 5% of the volume of the tissue biopsy sample. Therefore, a needarose to “microdissect” diseased cells from surrounding normal cells topermit molecular analysis of disease lesions in actual tissue.

[0193] To address this need researchers at the U.S. National Institutesof Health developed a technique known as “Laser Capture Microdissection”(“LCM”) for procuring pure cells from specific microscopic regions oftissue sections. See Emmert-Buck, et al., Science 274:998-1001,1996;Bonner, et al., Science 278:1481-1483,1997, incorporated herein in theirentirety. LCM allows small groups of cells to be isolated from tissuesections thereby allowing an investigator to collect only cells ofinterest so as to achieve high purity of the sample. Once collected,cells are homogenized and genomic DNA, total cellular RNA or totalproteins can be isolated. Details of LCM are described, for example, inPCT International Patent Applications publications WO 09917094A2 and WO098352A1, which are incorporated herein and are illustrated in FIG. 7.

[0194] In short, a laser beam focally activates a special transfer filmwhich bonds specifically to cells identified and targeted by microscopywithin the tissue section. The transfer film with the bonded cells isthen lifted off the thin tissue section, leaving all unwanted cellsbehind (which would contaminate the molecular purity of subsequentanalysis). This allows multiple homogeneous samples within the tissuesection or cytological preparation to be targeted and pooled forextraction of molecules and analysis.

[0195] In order to simplify the process of handling the transfer film,the film may be permanently bonded to the underside of a transparentvial cap, such as those available from Arcturus Engineering Inc.(Mountain View, Calif.). After the targeted cells are transferred to thecap surface the cap is placed directly onto a centrifuge tube to extractbiomolecules from the cap and purify biomolecules for subsequentanalysis, for instance using electrophoresis gels, DNA microarrays andthe like.

[0196] Unfortunately, many molecular biology assays such as Westernblotting are difficult to perform on LCM-collected samples since theamount of material collected per unit of time is very small. Whileanalysis of nucleic acids from LCM collected material is aided by theamplification techniques such as the polymerize chain reaction (PCR),protein amplification is not possible. Proteomics studies on LCMcollected samples are thus particularly difficult.

[0197] Another current limitation of LCM is that different cell subtypes(e.g. epithelium and connective tissue) must be transferred to differentcaps. Since the biomolecules (proteins and nucleic acid) are removedfrom the cap for further analysis, different cell types cannot be mixedon the same cap since it could not be determined from which cell type aparticular biomolecule originated. Thus users of LCM typically mustprocess a different cap for each cell type in a tissue section, aprocedure that is time consuming and creates variability in experimentaldesign.

[0198] Embodiments provided herein include methods and apparatuses fordetecting and analyzing biomolecules in a sample collected by LCM byeluting biomolecules away from the sample and binding them to one ormore membranes in a layered or stacked configuration, then visualizingthe biomolecules on the membranes.

[0199] In general, cellular samples embedded in/on an LCM transfer filmor the like are positioned adjacent to a stack of one or more membranes,and reagents and reaction conditions are provided so that biomoleculesare eluted from the cellular sample and transferred onto themembrane(s). Biomolecules on the membrane are then detected andvisualized using one or more detector molecules, for instance antibodiesor DNA probes having specific affinity for the biomolecules of interest.

[0200] There is illustrated in FIG. 8 a longitudinal section view of oneembodiment, preferably in the form of a kit, designated generally byreference numeral 58. Kit 58 generally comprises a membrane stack 13,LCM cap holder assembly 60, a pair of filter pads 24 and 26, and a fluidimpervious enclosure 28 such as a plastic bag or the like.

[0201] In some embodiments, membrane stack 13 comprises up to 20membranes, generally constructed as described herein. Representativemembranes 12 in stack 13 have a high affinity for proteins and otherbiomolecules, but have a low capacity for retaining such molecules. Inanother embodiment, a single membrane is used in lieu of a plurality ofmembranes. If only one membrane is used it need not have the lowcapacity requirements of certain other embodiments, and it can beconstructed of any of a variety of materials conventionally employed asblotting membranes, such as nitrocellulose or PVDF.

[0202] LCM cap holder assembly 60 is preferably constructed of a heatconductive material such as metal and has generally rectangulardimensions. A plurality of apertures 62 are defined by cap holderassembly 60 with each aperture adapted to receive a standard LCM cap 64such as those available from Arcturus Engineering, Inc. (Mountain View,Calif.) or a modified LCM cap 66 (FIG. 10). Mounted to caps 64 (or 66)is a standard LCM transfer film 68 having adhered thereto the selectedcellular material 70 that serves as the transfer sample 11 from thetissue sample following an LCM procedure. By way of example, LCM isperformed on tissue sections (such as frozen or fixed/paraffin embeddedsections) using the equipment such as that illustrated in FIG. 7according to known methods, such as those recommended by ArcturusEngineering, Inc.

[0203] First and second filter pads 24, 26 are preferably constructed ofa blotting paper such as GB004 Blotter Paper available from Schleicherand Schuell. Filter pads 24, 26 are saturated with a transfer buffersuch as Tris or phosphate base buffers.

[0204] Enclosure 28 may comprise any collapsible, fluid imperviousmaterial adapted to envelop the other kit components. Plastic bag 28 ispreferably a heat sealable pouch such as those available from KapakCorp. (Minneapolis, Minn.).

[0205] After microdissection, caps 26 can be stored frozen untiltransfer of the molecules is desired. Cellular material 70 embeddedwithin transfer film 68 is hydrated through gradient of ethanol andoptionally mildly digested with proteases. Caps 64 (or 66) are theninserted within apertures 62 defined in cap holder assembly 60 and thecap holder is placed adjacent to membrane stack 13 so that the transferfilm 68 is in direct contact with a membrane. First filter pad 24 isplaced above cap holder assembly 62 and second filter pad 26 is placedbelow membrane stack 13. (Both pads are soaked in a transfer buffer.)Pads 24 and 26, sandwiching the other components of the assembled stackof kit 58, are placed within enclosure 28. Most of the air is removedfrom enclosure 28 by gentle squeezing and/or vacuum suction and it issealed by a heat sealer such as the Impulse Sealer (AmericanInternational Electric).

[0206] With reference to FIG. 9 plastic bag 28 is placed between a pairof heating elements 56 such as those provided in Gel Dryers manufacturedby Bio-Rad Laboratories (Hercules, Calif.). The bag and its contents areheated to a temperature of between about 60 to 80° C., preferably toabout 70° C. for about two hours. Pressure is applied throughout theheating process using a weight 34, which may optionally be added as akit component.

[0207] In other embodiments, multiple caps are created from a singlecell type and the biomolecules (proteins and/or nucleic acids) aretransferred to the single membrane or membrane stack in the mannerdescribed herein. One membrane (or more) can then be cut into piecescorresponding to the number of caps so that the biomolecular contentfrom each cap may be separately incubated with a different detectormolecule or detection system.

[0208] It may be desirable to prevent rotation of the LCM caps duringthe transfer process so that positions of the cellular samples remainfixed relative to the membranes. This would be useful when particularregions of the film 68 are allocated to particular cell types (e.g.epithelium vs. connective tissue or diseased vs. normal cells). Bypreventing rotation of the LCM caps the user can correlate the positionof the biomolecules on the membranes with the region of film 68 and celltype from which the biomolecules originated. Lines or other indicia (notshown) may be provided on the membranes and caps 64 to aid the user inthis process. In order to prevent rotation, the standard LCM cap may bemodified as shown in FIG. 10. Modified cap 66 has a shank portion 72that defines a flat surface 74 (shown in FIG. 10B) that is adapted toengage an similarly shaped aperture in the LCM cap holder assembly (notshown). The size of cap 66 and corresponding transfer film may beenlarged so that cells of interest from an entire tissue section may bemicrodissected or otherwise transferred onto a single cap, therebysaving time and reducing experimental variability as compared to usingdifferent caps for each cell type as is the practice currently in use.

[0209] “Microarray” Transfer

[0210] Another use of the membrane arrays provided herein is to makemultiple copies of a cDNA or other microarray in a manner that is lessexpensive and labor-intensive than robotic systems. In particularembodiments, the plurality of DNA probes, antibodies, or combinationthereof, is applied to the stack of membranes from a plate having aplurality of wells (e.g., a microtiter or like plate), each containing adifferent DNA probe or antibody. The DNA probes or antibodies aretransferred from the wells to the stack so as to create a set ofsubstantially replicate microarrays.

[0211] With reference to FIG. 11, DNA sequences representing differentgenes are placed into individual microtiter wells 80 of a microtiterplate 82 (e.g. a 96-well plate). The microtiter plate 82 is placedadjacent to a stack 13 of membranes 12, to allow the contents of themicrotiter wells 80 to be transferred from the respective wells to thestack of membranes 13. In the illustrated embodiment, contents of thewells are transferred from the wells 80 to a top surface of the stack ofmembranes 13, so that the contents are applied in a pattern thatcorresponds to a pattern of the wells.

[0212] The DNA is transferred through the membranes in a direction ofmovement from the wells toward a wick member 84, and the spatialorientation of the samples is maintained. Because of the high affinity,low capacity characteristics of membranes 12, as the nucleic acidstraverse the capture membrane stack 13, a small percentage of DNAhybridizes to each membrane, creating a series of replicate copies, eachone containing a grid of DNA spots that match the orientation of the DNAsamples in the microtiter plate. Thus, a set of cDNA arrays may becreated in a very rapid and inexpensive fashion. Antibody and tissuelysate arrays can also be created by this method.

[0213] IV. Types of Samples

[0214] Any two-dimensional sample material that contains releasablebiomolecules can be used as a source of biomolecules in the providedtransfer processes. By “two-densional” it is meant that the material is,or can be formulated so that it is, substantially flat and relativelythin. Representative examples of substantially two-dimensional samplesinclude tissue samples such as thin section slices (e.g., archival orfrozen tissue samples), tissue arrays, cDNA or other nucleic acidmicroarrays, protein microarrays, 1-D protein gels, 1-D nucleic acidgels, 2-D protein gels, and so forth.

[0215] It is further contemplated that the described transfer methods,arrays, and devices can be used in forensic procedures to detect andstudy biological material such as bodily fluids; to detect biological(e.g., microbial) contamination of food or other substances; and soforth. In order to provide the sample in a substantially flat and thinformat, substances may be suspended in a liquid or gas, then run throughand optionally affixed to a filter such as a sheet of filter paper, withthe filter then used as the transfer sample. By way of example, a soilsample or fluid sample could be so prepared for transfer. Somesubstances may be compressed into a substantially flat form, forinstance by rollers or another spreading mechanism; by way of example, afood sample (e.g., ground meat) could be so prepared. Generally thesesamples can be referred to as structurally transformed samples, becausetheir format is altered to render them substantially two dimensionalprior to transfer onto a membrane stack.

[0216] Embodiments provided herein may be used to identify biomolecules(e.g., proteins or nucleic acids) in any biological sample includingbodily fluids (e.g. blood, plasma, serum, urine, bile, cerebrospinalfluid, aqueous or vitreous humor, or any bodily secretion), atransudate, an exudate (e.g. fluid obtained from an abscess or any othersite of infection or inflammation), fluid obtained from a joint, and soforth. Additionally, a biological sample can be obtained from any organor tissue (including or autopsy specimen) or may comprise cells.

[0217] V. Membranes

[0218] Also provided herein are membranes, which can be used in thedescribed methods of biomolecule separation.

[0219] In particular embodiments, the membranes comprise a material thatnon-specifically increases the affinity of the membranes to thebiological molecules (or a class of biomolecules such as proteins ornucleic acids) that are moved through the membranes. For example, themembranes may be dipped in, coated with, or impregnated withnitrocellulose, poly-L-lysine, or mixtures thereof. In certain examplesthe membranes are not treated with a material that blocks thenon-specific binding of the biomolecules to the membranes, at leastduring transfer of the biomolecules through the membranes. However, inother embodiments, some such blocking agents can be added to themembranes, as long as the amount of blocking agent minimizes the amountof biomolecules bound, without blocking it altogether. In certainexamples, blocking agent may be added to the membranes after transfer ofthe biomolecules through the membranes, but before or during exposure tothe detectors.

[0220] In particular examples, the membranes are sufficiently thin toallow th biomolecules to move through th plurality of membranes (forexample 10, 50, 100 or more) in the stack. Such membranes, for example,have a thickness of less than 30 microns. The membranes may be made of amaterial that does not substantially impede movement of the biomoleculesthrough the membranes, such as polycarbonate, cellulose acetate, ormixtures thereof.

[0221] The material of the membranes may maintain a relativerelationship of biomolecules as they move through the membranes, so thatthe same biomolecule (or group of biomolecules) move through theplurality of membranes at corresponding positions. In such examples,this coherence of relative relationships allows the different membranesto be substantial “copies” of one another, much like a “carbon copy”would be. However, like a “carbon copy” there may be some differencesbetween the different “copies” present in the different membranes.

[0222] In some embodiments, a membrane stack will include a number ofindividual membranes, for instance at least 2, at least 5, at least 10,at least 20, at least 50, or even more in some instances. Membranes inthe stack are generally constructed as described herein. Examples of themembranes are constructed of a porous substrate coated with a materialthat increases the affinity of the membrane to the biomolecules beingtransferred. The substrate may be constructed of polycarbonate or asimilar polymeric material that maintains sufficient structuralintegrity despite being made porous and very thin. Representativemembranes for use in the methods, devices, and apparatuses have a highaffinity for proteins and/or other biomolecules, but have a low capacityfor retaining such molecules. This binding profile permits biomoleculesto pass through the membrane stack with only a limited number beingtrapped on each successive layer, thereby allowing multiple “carboncopies” of the biomolecules in the sample to be generated. In otherwords, the low capacity allows the creation of multiple replicates asonly a limited quantity of the biomolecules is trapped on each layer.

[0223] With reference to FIG. 12, individual membranes 12 areconstructed of a porous substrate 90 coated with a material thatincreases the affinity of the membrane to the biomolecules beingtransferred. Substrate 90 is, for example, constructed of polycarbonateor a similar polymeric material that maintains sufficient structuralintegrity despite being made porous and very thin. However, in lieu ofpolycarbonate the substrate 90 may for example be constructed ofcellulose derivatives such as cellulose acetate, as well as polyolefins,(e.g., polyethylene, polypropylene, etc.).

[0224] The illustrated membrane 12 includes a coating 92 on its upperand lower surfaces to increase non-specific binding of the proteins orother targeted biomolecules. Although the binding to the coating is“non-specific” in the sense that it does not recognize particularproteins or other biomolecules, such as particular nucleic acids, it maybe specific in that it recognizes and specifically binds classes ofbiomolecules, such as proteins or nucleic acids, or combinationsthereof. Coating 92 in specific disclosed embodiments is nitrocellulose,but other materials such as poly-L-lysine may also be employed.

[0225] Before being applied to substrate 90, the nitrocellulose isdissolved in methanol or other appropriate solvent in concentration from0.10%-1.0%. The membranes are immersed in this solution as describedmore fully in the Examples, below. In lieu of coating 92, nitrocelluloseor other materials with an affinity for biomolecules can be mixed withthe polycarbonate before the substrate is formed thereby providing anuncoated substrate having all of the desired characteristics of themembrane. Alternative coating methods known in the art may be used inlieu of dip coating including lamination. Alternatively, only onesurface may be coated, such as the surface that faces the sample,instead of both surfaces.

[0226] It is a particular feature of certain embodiments that membranes12 have a high affinity for proteins and other biomolecules, but have alow capacity for retaining such molecules. This feature permits themolecules to pass through the membrane stack with only a limited numberbeing trapped on each of the successive layers thereby allowing multiple“carbon copies” to be generated. In other words, the low capacity allowsthe creation of multiple substantial replicates as only a limitedquantity of the biomolecules are trapped on each layer. If a membranewere used that had a high binding capacity for biomolecules-such as withnitrocellulose membranes conventionally used with gel blotting-multiplereplicas could not as easily be made. More specifically, the affinityand capacity of membrane 12 should be such that when at least five andpreferably more than ten membranes are stacked and applied to a sampleaccording to a disclosed method, most of the biomolecules of interestcan be detected on any and all of the membranes, including thosepositioned furthest from the sample.

[0227] With reference to FIG. 13, the aforementioned technique may bedescribed as “direct capture” since the target biomolecules 100 arecaptured directly on the surface of membranes (or within the membrane),instead of being captured indirectly by a binding agent (such as anantibody or nucleic acid probe) applied to the membrane. During thisdisclosed process different components of the sample bind to themembrane with the same affinity, but directly proportional to theirconcentration in the sample (a component with a higher concentrationwill leave more molecules on each membrane, and a component with a lowerconcentration will leave less molecules on each membrane). A detectormolecule 104, such as a labeled antibody that specifically binds to thebiomolecule 100 at illustrated epitopes 102, may be utilized to detectbiomolecule bound to the membrane. In examples in which the amount of acomponent bound to the membrane is proportional to the amount of thecomponent in the sample, an amount of the detector molecule can becorrelated to an amount (or relative amount) of the biomoleculedetected.

[0228] In order to achieve high affinity and high capacity for aparticular group of biomolecules from a sample, coating of the membraneswith a captor molecule 106 is performed in the method described byEnglert et al. (Cancer Research 60:1526-1530, 2000). This may bereferred to as “indirect capture” and is illustrated in FIG. 14. Captor106 can be cDNA, antibody, or any other protein specific for the targetof interest. Single-stranded cDNA molecules generated by number of means(Polymerase Chain Reaction, nick translation, reverse transcription,oligonucleotide synthesis) or proteins (like immunoglobulin) can bedirectly attached to the membrane. Alternatively, the linker-arms thatwould allow spatial control of the captor binding could be directlyattached to the membrane followed by captor attachment to them. Thiswill expose the majority of the active target recognition sitesincreasing that way capacity of the indirect capture. Streptavidincoated membranes may be employed to bind end-biotinylated nucleic acidsand randomly biotinylated proteins, or protein A and protein G to bindimmunoglobulins.

[0229] In another embodiment (illustrated in FIG. 15), each of themembranes 108 comprise a ligand coating (e.g., a unique ligand coating,in that it is different from the others in the stack) that selectivelybinds to proteins in the biological sample based on a particularcharacteristic of the protein chemistry (e.g. hydrophobicity,carbohydrate content, etc.) As a result, the membranes 108 function tofractionate the proteins rather than replicate them as with membranes 12in other described embodiments. The coating could be made in manydifferent ways so that each membrane binds a selective subset of thetotal protein content in the sample. For example, carbon chains ofincreasing length, starting with a small carbon molecule can be used inthe coating. As the number of carbons increases the ability to bind toproteins increases. Thus, for example, the first layer may have a sixcarbon-chain coating and will only bind to the most hydrophobic proteinsin the sample, the remaining proteins will pass through to the nextlayer; the second layer has an eight-carbon chain and will pull outslightly less hydrophobic proteins while the remaining proteins passthrough; the third layer has a ten carbon-chain, etc.

[0230] Thus, with another embodiment, each of the membranes will bind toa different group of proteins essentially permitting “3-D gelelectrophoresis” by allowing proteins to be separated into threedimensions: in the X and Y dimensions by charge and mass, and then inthe Z dimension by an additional chemical characteristic. The proteinson the membranes according to the second embodiment can be visualized bythe immuno-staining and imaging methods set forth below. They may alsobe advantageously analyzed by mass spectrometry either withoutadditional cleavage or after such cleavage (see, e.g., WO00 045168), orby other means. Examples of the methods and kits facilitate suchanalysis because the stratification by the different membranes helps toexpose moderate and low abundance protein spots that would otherwise beundetectable on standard 2-D gels. The more spots that are available foranalysis, the more data can be generated by mass spectroscopy or by suchother approaches.

[0231] Other Membrane Characteristics

[0232] It is a particular feature of some embodiments that membranesused for the transfer have a high affinity for proteins and/or otherbiomolecules, but have a low capacity for retaining such molecules. Thisfeature permits the molecules to pass through the membrane stack withonly a limited number being trapped on each of the successive layers,thereby allowing multiple replicate “carbon copies” to be generated. Inother words, the low capacity of the membrane material allows creationof multiple replicates, since only a limited quantity of thebiomolecules (e.g. proteins) are trapped on each layer.

[0233] More specifically, in specific embodiments the affinity andcapacity of membrane should be such that when at least five andpreferably more than ten membranes are stacked and applied to a sampleaccording to one of the provided methods, most of the biomolecules ofinterest can be detected on any and all of the membranes, includingthose positioned furthest from the sample. If a membrane were used thathad a high binding capacity—such as the transfer membranes used withconventional gel blotting, multiple replicas could not be made in thismanner unless the binding capacity of the membrane was overwhelmed bythe amount of biomolecule applied to the membrane.

[0234] To maintain the binding capacity of membrane sufficiently low toavoid trapping of too much of the sample, the thickness of the substrateis, for example, less than about 30 microns, and in particularembodiments is between about 420 microns, for example between about 8 to10 microns. The pore size of the substrate is, for example, betweenabout 0.1 to 5.0 microns, such as about 0.4-0.6 microns, and morespecifically 0.4 microns. Another advantage of using a thin membrane isthat is lessens the phenomenon of lateral diffusion. The thicker theoverall stack, the wider the lateral diffusion of biomolecules movingthrough the stack.

[0235] It will be appreciated that because the size of the membranes inthe stack/array can be varied, the user has the option of analyzing alarge number of different samples in parallel, thereby permitting directcomparison between different patient samples (e.g. different patientsamples, or patient samples and a reference standard, or samples ofdifferent tissues or species, etc.). For example, different samples fromthe same patient at different stages of disease can be compared in aside-by-side arrangement, as can samples from different patients withthe same disease. By way of alternative example, the area of proteinseparation resulting from most 2-D gels is generally between about 10×10cm to 20×20 cm; membranes used for transfers of 2-D gels may varyaccordingly.

[0236] Membrane Construction

[0237] The membrane substrate includes a coating on its upper and lowersurfaces to increase specific binding of the proteins or other targetedproteins. The coating is preferably nitrocellulose but other materialssuch as poly-L-lysine may also be employed. Before being applied tosubstrate, the nitrocellulose is dissolved in methanol or otherappropriate solvent in concentration from 0.1%-1.0%. The membranes areimmersed in this solution as described more fully in the Examples,below. In lieu of coating, nitrocellulose, or other materials with anaffinity for proteins, can be mixed with the polycarbonate before thesubstrate is formed thereby providing an uncoated substrate having allof the desired characteristics of the membrane. Alternative coatingmethods known in the art may be used in lieu of dip coating, includinglamination. In all instances it should be understood that preferablyonly one surface—the surface that faces the sample—is coated or treated,instead of both.

[0238] Framed Membranes

[0239] In another embodiment, with reference to FIGS. 16-18, framedmembrane stack 110 comprises a plurality of individual membrane units112 releasably secured to one another. Each membrane unit 112 comprisesa membrane 12 having a frame 114 mounted about the periphery thereof.Membrane unit 112 can vary in size but should be large enough so thatmembrane 12 can overlay a typical electrophoresis gel.

[0240] The number of membrane units 112 included in stack 110 can varydepending on the number of proteins to be detected from the gel. Formost applications, from 3 to 25 or more membranes will be sufficient,preferably from 5 to 15 and most preferably about 10 to 12. The entirethickness, Ts, of stack 110 (FIG. 16) is in some embodiments no morethan about 0.25 cm.

[0241] In some embodiments, in order to give each membrane sufficientrigidity to enable it to be separated the other membranes in stack 110and individually processed, a frame 114 is mounted onto the periphery ofmembrane 12 thereby forming membrane unit 112. Frames 114 preferablycomprise a generally “Z” shaped configuration covering three sides ofthe membranes while defining an open space or gap 120 that functions asa channel to permit the manual removal of air pockets or fluids in themanner described.

[0242] The composition and dimensions of frame 114 should be such thatthe frame provides sufficient rigidity for the user to grip the framewith one hand and manipulate the membranes as needed. At the same time,the frames must be sufficiently thin so that when stacked they do notinterfere with protein transfer from the gel onto the membrane stack110. Each membrane 12 in stack 110 should be capable of making directcontact with adjacent membranes during the transfer process describedherein.

[0243] The width W (FIG. 18) of frame 114 is preferably between about0.3 to 0.7 cm and the thickness of the frame, Tf, is between about 0.005to 0.03 cm., most preferably about 0.01 cm thick. Thus, frame 114 isabout ten times thicker than membrane 12. In certain embodiments, thematerials that comprise frames 114 are able to maintain their structureand function at temperatures of up to 80° C. but are able to melt whenapplied to a typical heat-sealing apparatus. One skilled in the relevantart will readily appreciate that a variety of compositions andconfigurations of frames 114 could meet these requirements. Examples ofmaterials that may be employed to make frames 114 are transparency filmavailable from Canon or any thin plastic sheet made of polycarbonate,polyester, polyvinylchoride or polyvinilechloride.

[0244] As best viewed in FIG. 17, a pair of outwardly depending tabs 116is defined by frame 114. Each tab is adapted to be sealed to thecorresponding tab on an adjacent membrane so as to hold stack 110together during the gel transfer process. After the proteins aretransferred onto the membranes tabs 116 are cut with a scissors so thatthe membranes may be separated and incubated in separate detectionsolutions.

[0245] At least one side of frame 114 defines a surface 118 upon whichindicia may be imprinted. The indicia may include the name of theproduct or manufacturer or the membrane number. Machine-readable indiciasuch as a bar code or the like (not shown) may also be provided.

[0246] Frames 114 may be mounted to the perimeter of membranes 12 byvarious means readily familiar to those skilled in the art including useof adhesives such as rubber cement or 3M adhesive or conventionalheat-sealing or laminating techniques.

[0247] VI. Analysis of Membrane Replicates

[0248] After transfer, the processed membranes (or layers) can beseparated and each incubated with one or more different detectormolecules (such as nucleic acid hybridization probes, lectins, orantibodies) specific for particular targets of interest. In certainembodiments, the detectors/probes employed are labeled or otherwisedetectable using any of a variety of techniques such aschemiluminescence. Thus, while each membrane has essentially the samepattern of biomolecules bound to it, different combinations ofbiomolecules can be made observable on each membrane by selectingparticular probes to be applied and detected.

[0249] By way of example, one membrane layer may display proteinsinvolved in programmed cell death (apoptosis) while an adjacent layermay display enzymes involved in cell division such as tyrosine kinases.

[0250] In addition to proteins, nucleic acids may be targeted anddetected by using labeled DNA hybridization probes rather thanantibodies. Moreover, both protein and nucleic acid targets can bedetected in parallel by applying both antibodies and nucleic acid probesto different layers of the stack. Similarly, carbohydrates can bedetected using carbohydrate-binding molecules such as lectins.

[0251] Digital images of membranes may be created using a variety ofinstruments including the Image Station® CCD instrument available fromKodak Scientific Imaging (New Haven, Conn.). Alternatively images may becaptured on film (such as X-ray film) and digitalized by flat bedscanners. Software is preferably provided to align the images andperform densitometry functions. The user can select the region ofinterest for analysis and the signal intensities are recorded andnormalized. The numerical intensity values are then compared.

[0252] For analysis of transferred proteins, after the transfer by anyof the herein-described protein-transfer techniques, the membranes areseparated from stack and each is incubated in a separate solution ofprimary antibody specific for a desired protein. Only the bandcontaining this protein binds the antibody, forming a layer of antibodymolecules. After incubation for about 1-8 hours, the membranes areusually washed in buffer to remove unbound antibody.

[0253] For detection of the proteins on the membranes (in the form ofbands, spots, or “in situ” from tissue transfers), the loaded membranesare incubated in a secondary antibody that binds to the primaryantibody. The secondary antibody may be covalently linked to an enzymesuch as horseradish peroxidase (HRP) or alkaline phosphatase (AP) thatcatalyzes substrate and protein/antibody complex can be visualized usinga number of techniques such as ECL, direct fluorescence, or calorimetricreactions. ECL is preferred. Commercially available flatbed scanners maybe employed in conjunction with film. Alternatively, specialized imaginginstrumentation for ECL, such as the Kodak IMAGE STATION available fromNEN may be utilized and digital imaging software can be employed todisplay the images according to the preference of the user.

[0254] In lieu of antibodies, other ligands may be employed asdetectors. Ligands can be antibody fragments, receptors, receptorligands, enzymes, viruses or viral particles, enzyme substrates or othersmall molecules that bind to specific proteins. Moreover, in addition toidentifying proteins of interest structurally, kits can also be employedto identify the functional state of proteins. One way to do so is to usephospho-specific antibodies to determine the phosphorylative state ofprotein(s) of interest. Another approach to identifying protein functionis to first renature the proteins on the membranes by any of a number oftechniques known in the art such as incubating the membrane in Triton-X®(octylphenol polymerized with ethylene oxide). Once renatured, proteinswill regain their enzymatic activity and one of several substratedegradation assays known in the art can be used. With this approach theactivity of kinases, phosphates and metalloproteinases can bedetermined.

[0255] Panels for scientific research may be grouped by the proteinsinvolved in a particular cellular phenomenon such as apoptosis, cellcycle, signal transduction, etc. Panels for clinical diagnostics may begrouped by proteins associated with a particular disease such asAlzheimer's disease, prostate cancer, etc.

[0256] In many embodiments, the detectors/ligands employed are labeledor otherwise made detectable using any of several techniques, such asenhanced chemiluminescence (ECL), fluorescence, counter-ligand staining,radioactivity, paramagnetism, enzymatic activity, differential staining,protein assays involving nucleic acid amplification, etc. The membraneblots are preferably scanned, and more preferably, digitally imaged, topermit their storage, transmission, and reference. Such scanning and/ordigitalization may be accomplished using any of several commerciallyavailable scientific imaging instruments (see, e.g., Patton et al.,Electrophoresis 14:650-658, 1993; Tietz et al., Electrophoresis12:46-54, 1991; Spragg et al., Anal Biochem. 129:255-268, 1983; Garrisonet al., J Biol. Chem. 257:13144-13149, 1982; all herein incorporated byreference).

[0257] Example Detection Chemistries with Detector Cocktails

[0258] In certain embodiments, after proteins have been transferredthrough the membrane stack, individual membranes layers are separatedand each is incubated in a separate antibody (or other detectormolecule) cocktail. A key advantage of creating multiple replicate blotsis that many more detector molecules (e.g., antibodies) can be usefullyemployed than if all of the detectors had to be crowded onto a singleblot.

[0259] An exemplary process for designing the ligand cocktails—and fordetermining which proteins will be identified on each membrane layer—isprovided below. First the panel of proteins of interest is selected.These can be randomly selected proteins and/or proteins that are notdirectly related to one another or may be groups of known proteinspreviously implicated to play a role in one or more particular cellularphenomena (e.g. apoptosis, cell cycle progression) or a particulardisease (e.g. prostate cancer specific antigen, PSA). These should beproteins that have been characterized by sequence or coordinates on 2-Dgels or for which ligands have been or could be generated. Data bases ofannotated 2-D gels include the Quest Protein Database Center(http://siva.cshl.org), the Swiss 2-D PAGE database(http://expasy.cbr.nrc.ca/ch2d), Appel et al. Electrophoresis. 14(11):1232-1238, 1993; the Danish Centre for Human Genome Research(http://biobase.dk/cgi-bin/celis), Celis et al., FEBS Lett.398(2-3):129-134, 1996, etc. Antibodies may be obtained from a varietyof sources such as BD Transduction Laboratories (Lexington, Ky.) orSanta Cruz Biotechnology (Santa Cruz, Calif., USA).

[0260] Although, as discussed above, any of a broad class of ligands maybe employed, for simplicity the embodiment is illustrated with referenceto the use of antibody ligands. Immunological identification of theproteins on the membranes thus preferably involves the selection ofantibodies having a high affinity and specificity for their targets.However, antibodies, both monoclonal or polyclonal, frequently recognizemore then one protein in Western blotting detection. Thiscross-reactivity phenomenon becomes increasingly apparent as theconcentration of antibody increases relative to that of the sampleproteins. Hence, the first step in the antibody selection processpreferably involves choosing antibodies (and their workingconcentrations) that consistently visualize preferably 1 but no morethen 5 proteins on the same membrane. When the detector antibody bindsto more than one spot, the undesired proteins (“false spots”) can beeliminated based on their X-Y positions on the membranes. Since themolecular weight and charge (pI) of a given protein is generallyconstant, it should appear at about the same coordinates on the gel eachtime it is run.

[0261] If two or more proteins in a sample are of similar size andcharge—and therefore migrate to the same general vicinity on thegel—they would likely create overlapping spots if detected on the samemembrane. In a preferred embodiment, examples of the method avoid thisproblem by designing the antibody cocktail to detect adjacent oroverlapping proteins on different membranes.

[0262] The cocktail design process can be readily understood withreference to the following hypothetical example (illustrated in FIG.19). For simplicity in this example, thirteen proteins annotated as A-Min FIG. 19A are sought to be identified using only a three-layermembrane stack. The ligands employed in the example are antibodies, andthree cocktails, one for each stack, each with four to six differentantibodies, are employed.

[0263] For the first membrane cocktail (corresponding to layer one)antibodies are screened for protein spot A and the most specificantibody is selected. Antibodies for spots B-E are picked the same way.Because spots F and G overlap with spot E these are put aside for otherlayers. The second and third cocktails (corresponding to membrane layerstwo and three) are created using the same considerations: (1) if thespot position generated by any two antibodies cannot be easilydistinguished, the antibodies will not be used in the same cocktail; (2)if any antibody results in a background spot near-the spot generated byanother antibody, the two antibodies will not be included in the samecocktail unless the background spot is remote from other spots on thatlayer (e.g. spots B and D on layer 2 created due to cross-reactivityfrom antibodies directed to other spots), in which case suchcross-reactivity is simply ignored when the membrane spots are comparedto the template. Applying these considerations to the hypotheticalexample results in three cocktails corresponding to the layersillustrated in FIGS. 19B-D.

[0264] Once assembled, the antibody cocktails will be additionallytested for their specificity by two different control tests. In a firsttest, membranes made from the transfer of a single gel (or from severalgels that contain the same sample and were prepared in the same manner)will be probed with cocktails that differ in only one antibody component(each cocktail will lack one of the antibodies). As a result of thisprocedure, immunoblotted membranes should differ from each other in onlyone spot. In a second test, antibody cocktail will be incubated for0.5-12 hours at 4-25° C. with a mixture of epitopes (peptides orproteins) that are used for immunization. During this incubation, freeantibodies bind to the appropriate epitopes and become immobilized andfunctionally inactive. Since the cocktail becomes depleted of freeantibodies subsequent incubation of the membrane with this free antibodydepleted mixture should yield no specific signal.

[0265] Each cocktail will also include one or more antibodies against“housekeeping” proteins (i.e., abundant structural proteins found in alleukaryotic cells such as actin, tubulin, etc.). Thus, for example, theantibodies employed with respect to membrane Layer #1 of FIG. 19 willcontain an antibody to actin, which will result in the production of aspot. These antibodies serve as internal landmarks to normalize samplesfor loading differences and to compensate for any distortion caused bygel running process. Once the cocktails are designed, they can be reusedin any kit that seeks to identify the same panel of proteins that wereidentified in creating the cocktails, regardless of the origin of thesample.

[0266] It will be appreciated that the present disclosure allows notonly the simultaneous characterization of a large number of differentproteins but also permits the characterization of a large number ofcharacteristics of a single protein based on number of differentcharacteristics. For example, the protein p70 S6 kinase, required forcell growth and cell cycle progression, is activated by phosphate groupattachments (phosphorylation) to threonine on position 229 and/or 389 ofthe protein. Identification of this kinase would provide not only adetermination of its presence or absence but also a demonstration of itsactivity. By way of example, with a kit containing at least afour-membrane stack, four copies can be made of a 2-D gel. The firstmembrane would be incubated in antibody specific for the whole proteinto determine if this enzyme is present in the sample or not The secondmembrane can be used in kinase assay to determine if the enzyme isactive or not. The third membrane can be probed with phospho-p70 S6kinase (Thr229) antibody to determine if activity of the enzyme is dueto activation of this site. The fourth membrane can be probed withphospho-p70 S6 Kinase (Thr389) antibody to determine if the activity ofthe enzyme is due to activation of that site. And since all of thesetests are done on the single sample (rather than different batches ofthe same sample) the information obtained is more reliable.

[0267] Antibody cocktails (such as those illustrated in FIGS. 4 and 5,reference number 38) are preferably stored in vials, preferably made ofplastic or glass, and are optionally combined in a kit to create a“panel” of protein targets of interests. Panels for scientific researchmay be grouped by the proteins involved in a particular cellularphenomenon such as apoptosis, cell cycle, signal transduction, etc.Panels for clinical diagnostics may be grouped by proteins associatedwith a particular disease such as Alzheimer's, prostate cancer, etc.

[0268] VII. Kits

[0269] Other embodiments of the disclosure include kits that contain amembrane array for detecting biomolecules (such as proteins or nucleicacids) in a sample. The array includes a plurality of membranes, each ofwhich has a non-specific or substantially same affinity for thebiomolecules. Certain provided kits also include one or more containersof detector molecules, such as antibodies or probes (or mixtures ofantibodies, mixtures of probes, or mixtures of the antibodies andprobes), for detecting biomolecules captured on at least one of themembranes. In particular examples of the kit, the membranes are polymersubstrates containing or coated with a material (such as nitrocellulose)for increasing an affinity of the substrate to the biomolecules.

[0270] Kits may additionally contain reagents for effecting thedetection of detector/ligand-biomolecule binding, buffer, and/orinstructions or labels that indicate the particular detector or detectorcocktail to be applied to a particular membrane. Software such as thatdiscussed herein may also be included in the kit or may be accessiblevia modem, the Internet, by mail, or by other means.

[0271] Primary antibodies to particular groups of proteins, such asbiochemical pathways may be optionally included with a kit.Alternatively the user can supply primary antibodies.

[0272] The methods and kits allows up to several thousand discreteprotein spots to be identified, annotated, and, at the user's option,compared to the pattern of protein spots generated from other biologicalsamples stored in a database.

[0273] Certain kit embodiments have been discussed above, includingfirst kit 36 and second kit 58. Also provided is another specificembodiment, directed to a method and a kit 122 for identifying (i.e.detecting, annotating, and/or characterizing) groups of proteins thathave been separated by gel electrophoresis. As illustrated in FIG. 20,representative kit 122 comprises the following components: (i) amembrane stack 13 or framed membrane stack 110 (as illustrated) uponwhich the proteins are transferred, (i) protein transfer reagent(s) 124and (iii) protein detector molecules, such as stain 126 andprotein-specific detector molecule 128. The kit may also includesoftware 46 (not shown in FIG. 20) that allows the user to analyze andmanipulate the images produced so as to yield a “proteomic image” of thebiological sample being tested and compare it to proteomic images fromother samples in a database. Alternatively the software may be acquiredor accessed independent of the kit.

[0274] In some embodiments, transfer reagent is also provided with akit. Examples of transfer reagents include Tris, Phosphate, Tris/Glycineor Phosphate/Glycine buffers with an alkaline pH (e.g., 8.0-9.5), withor without methanol (usually 20% or less) and/or SDS (in someembodiments 0.05% or less, and in particular embodiments 0.025% orless). Specific examples of transfer reagent suitable for use inexamples of such kits are in the Examples.

[0275] In addition to identifying proteins of interest structurally,kits are provided that can be employed to identify the functional stateof proteins. One way to do so is to use phospho-specific antibodies todetermine the phosphorylation state of protein(s) of interest. Anotherapproach to identifying protein function is to first renature theproteins on the membranes by any of a number of techniques known in theart (such as incubating the membrane in Triton-X-100® (octylphenolethylene oxide condensate). Once renatured, some proteins will regaintheir functional activity and one of several substrate degradation ormodification assays known in art can be used. With this approach theactivity of kinases, phosphates and metalloproteinases, etc., can bedetermined.

[0276] VIII. Devices and Apparatuses

[0277] In certain provided embodiments, particularly those which employcontact transfer, the transfer can be effected by placing the assembledmembrane stack into a gel drier-type apparatus, which applies heatand/or pressure to the stack. The combination of heat and pressure beingapplied causes biological components, including proteins and/or nucleicacids and/or carbohydrates and/or lipids, to be transferred from thesample 11 to membrane stack 13. This produces multiple copies orreplicas of the biomolecular content of the tissue sample, due at leastin part to the binding characteristics of the membranes.

[0278] In lieu of gel dryers, a specialized instrument 130 (FIG. 21) maybe employed to provide heat and/or pressure to the sample and membranestack. The instrument comprises a body 134 and a lid 136, each having aface 132 a, 132 b which serves as one of the substantially flat surfaces132. The surfaces may be provided by the upper face 132 b of the body134 and the lower face 132 a of the lid 136 directly, or may be providedby a substantially flat panel or other flat object disposed on a face132 a, 132 b of the body 134 or lid 136.

[0279] One or both of the substantially flat surfaces may protrude inorder to ensure adequate contact to provide pressure between them. Inthe illustrated embodiment, for instance, the upper substantially flatface 132 a is a surface of a member that protrudes from th lower face132 a of the lid 136. In some embodiments, one or the other or both ofthe substantially flat surfaces 132 a, 132 b may be compressible (forinstance, somewhat spongy), to further ensure that pressure applied tothe sample and membrane stack is relatively complete and even across thesurface of the stack.

[0280] The lid 136, in some provided embodiments including thatillustrated in FIG. 21 may be of sufficient weight to provide sufficientpressure to a sample and membrane stack placed under the lid 136 so thatit facilitates biomolecule transfer as described herein. Such weight isnot required, but in those embodiments wherein the lid 136 does notprovide sufficient weight, another mechanism for applying pressure isincluded. Such means includes for instance a separate weight (notshown), such as a weight 34 placed on the upper surface of the lid 136,or clips, springs, clamps or the like that urge the lid 136 toward thebase 134 with sufficient force to provide the amount of pressure neededto facilitate transfer.

[0281] In some embodiments, the lid 136 may be hingedly attached to thebody 134, such that when the lid 136 is lifted it does not come fullyaway from the body 34 but remains connected in at least one place. Inthe illustrated example (FIG. 21), two hinges 138 are provided tomaintain the connection between the body 134 and the lid 136. Inparticular embodiments, the hinge or hinges 138 are “loose” or“floating,” in that they permit some play between the lid 136 and thebody 134. This play permits the device to accommodate assembled contacttransfer stacks of different thickness, while still adequately applyingsufficient and even pressure to the stack. Though some embodiments arelarge enough to accommodate multiple stacks in side-by-side arrays, itis not recommended that stacks of substantially different thickness betransferred in the same device at the same time, as the applied pressuremay not be adequate on thinner stacks when a substantially thicker stackis present between the faces 132 a, 132 b.

[0282] Some embodiments of the device 130 are capable of supplying heatas well as pressure to the contact transfer stack. These embodiments maycontain, for instance, a heater element (not shown) in the body 134 orthe lid 136, or both, that provides heat to one or both of thesubstantially flat faces 132 a, 132 b. Examples of such heated devices130 will be equipped with an internal or external power source, forinstance a battery (not shown) or connection to a source of alternatingcurrent (not shown). Methods of and mechanisms for providing heat to asurface are well known, as are thermostats for controlling the level ofheat provided. Specific examples of heated devices 130 will include amechanism for controlling whether or not heat is generated (e.g. an“ON/OFF” switch 140 as shown in FIG. 21), a mechanism for regulating thelevel of heat produced (e.g., a thermostat, with or without a usercontrol), and/or an indicator that indicates when the device is heatingor heated. In the illustrated embodiment, an indicator light 142 isprovided, which is capable of indicating when the device reaches afactory-set temperature (e.g., 80° C.), and is thus ready for use.

[0283] Specific examples of the heated device 130 that include a heaterelement in both the lid 136 and the body 134 may include a mechanism orcontrol (such as dial 144) for selecting whether one, the other, or bothheater elements are engaged when the device is turned on.

[0284] IX: Applications

[0285] The heat and pressure applied to contents of the enclosure permitproteins and other molecules to be transferred from the embeddedcellular material to the membrane stack. This produces multiple copiesor replicas of the biomolecular content of the cellular sample. Theprocessed membranes (or layers) are then separated and each is incubatedwith one or more different probes or antibodies specific for particulartargets of interest. The probes employed are labeled or otherwisedetectable using any of a variety of techniques such aschemiluminescence. Thus, while each membrane has essentially the samepattern of proteins bound to it, different combinations of proteins aremade visible on each membrane due to the particular probes or antibodiesselected to be applied. For example, one membrane layer may displayproteins involved in programmed cell death (apoptosis) while an adjacentlayer may display enzymes involved in cell division such as tyrosinekinases. In addition to proteins, nucleic acids may be targeted by usinglabeled DNA probes in lieu of antibodies. Moreover, both protein andnucleic acid targets can be detected in parallel by applying bothantibodies and probes to different layers of the stack. Commerciallyavailable flatbed scanners and digital imaging software can be employedto display the images according to the preference of the user.

[0286] With reference to FIG. 20, kit 122 may be used to identifyproteins that have been separated on electrophoresis gels, bothtwo-dimensional gels and one-dimensional gels. Proteins are isolatedfrom a biological sample and separated on the gel according totechniques well known in the art, such as those described herein and inManabe, Electrophoresis. 21(6):1116-1122, 2000; Oh et al.,Electrophoresis. 20:766-774, 1999; Dunn, J Chromatogr. 418:145-185,1987,

[0287] In some embodiments, after gel 50 is run, it is removed from theelectrophoresis apparatus and sandwiched and placed in a transferapparatus such as the type typically used in creating Western blots.Such devices are available, for example, from Biorad Laboratories, Inc.,Novex, Inc. and Amersham Pharmacia Membrane stack 13 is positionedbetween the electrodes adjacent to gel 50 as illustrated in FIG. 22.While only about a half-dozen membranes are shown in FIG. 22 it will beappreciated that up to one hundred may be employed depending on thenumber of targets sought to be identified in a panel, the quantity ofproteins present in the sample, and the thickness of the materialemployed to construct membranes 12. Optionally, membranes 12 may bepackaged in a suitable sealed enclosure or frame (not shown) to maintaintheir integrity and prevent contamination. Sponge pads 130, preferablyconstructed of foam, rubber or filter paper and layers of filter paper14 are also sandwiched between the electrodes as shown in FIG. 22.

[0288] Transfer buffer (25 mM Tris pH 8.3, 192 mM glycine, 0.025% SDSand 20% methanol) is applied to elute and transfer proteins from the gel50 to the membranes 12. Any of a variety of conventional methods foreffecting such transfer may be employed, including wet tank transfer,and semi-dry transfer. In a wet tank transfer, the membranes areimmersed into a tank containing buffer, in a semi-dry transfer, themembranes are blotted with moist pads. In both cases, the membranes aresubjected to a voltage potential (e.g., 125-150 mAmps for 1-10 hours).In such transfer, it is important that a tight contact be createdbetween the membranes and the gel. Wet tank transfer is preferred. For amembrane of 10×10 cm², a tank containing 400-500 ml of buffer may beemployed. Preferred transfer conditions are 60-110 mAmps for 1-2 hours.

[0289] After transfer the membranes are separated and incubated withdetector antibody(s). Antibodies are selected based on the types oftarget molecules sought. Membranes are washed in a buffer, and theprotein/detector complex can be visualized using a number of techniquessuch as ECL, direct fluorescence, or colorimetric or calorimetricreactions. Commercially available flatbed scanners may be employed inconjunction with film, to detect or record signals. Alternatively,specialized imaging instrumentation (e.g., for ECL), such as the KodakIMAGE STATION (NEN) may be utilized. Digital imaging software can beemployed to display the images according to the preference of the user,as discussed herein.

[0290] In addition to use with 2-D gels, provided methods may beemployed to identify proteins that have been separated by a 1-D gel suchas conventional gels for separating proteins by size, and gel shiftassays. Gel shift assays (also known as “mobility shift assays”) are themost commonly used tool for studying protein—DNA interactions. The assayis based on labeling of the DNA fragment that contains presumptiveprotein binding site and incubation of that labeled fragment withprotein that binds to that site. If they interact, complex will beformed. If source of protein is a cell extract (rather than a solutionof in vitro synthesized proteins), the complex may contain number ofproteins, of unknown identity, that interact with each other. Afterbinding, a mixture of DNA and proteins is loaded onto a non-denaturingpolyacrylamide gel and the proteins are separated based on their size.DNA-protein complexes are visualized by exposure to X-ray film, or byother means. The higher the bands are in the gel, the larger the size ofthe DNA-protein complex. In most cases, this type of analysis does notreveal identity of the protein(s) in the complex.

[0291] As illustrated in FIG. 23, membrane stack 110 may be used toidentify biomolecules that have been separated on electrophoresis gels,including proteins that have been separated on one-dimensional (1-D)gels 132 or two dimensional (2-D) gels (such as 50, not shown in thisfigure) as well as nucleic acids that have been separated on agarosegels. The following description relates to use of embodiments inconjunction with protein detection of 1-D gels.

[0292] Proteins are isolated from a biological sample and applied andseparated onto a gel 132, typically a sodium dodecylsulfate—polyacrylamide gel, which is cast, for example, as a square slabgel with a thickness between 0.5 to 2.0 mm. Pre-cast gels useful withthe present disclosure can be obtained from a variety of suppliersincluding InVitrogen (Carlsbad, Calif.).

[0293] Unlike with conventional blotting, wherein less than 30micrograms of protein is loaded into each well of the gel, according tospecific methods herein between about 50 to 100 micrograms of protein isloaded into each well. The amount loaded will depend upon the number of“copies” of membranes to be created and size of the protein one wishesto detect (see Example 11).

[0294] In some embodiments, the components of transfer buffer 124 areprovided in separate containers, which are combined and applied to eluteand transfer proteins from the gel 132 to the membranes 12. About 500milliliters may be used in each transfer, with average length of thetransfer being about 1-2 hours. With reference to FIG. 23, 24, and 6,separated proteins on gel 132 are transferred to framed membrane stack110 (though a stack of unframed membranes could be used) by one ofseveral alternative techniques.

[0295] A first technique, illustrated in FIG. 24, involves electrictransfer using a standard gel electro-blotting apparatus such as theMiniCell unit (Bio-Rad Laboratories, Calif.). Gel 132 is removed fromthe electrophoresis apparatus and placed adjacent to membrane stack 110.The gel 132 and membrane stack 110 are then placed between the anode 134and cathode 136 of electro-blotting apparatus 138 with sponge pads 130positioned as shown. The electro-blotting apparatus 138 is activatedwith a voltage of about 59-63 volts for about 60-70 minutes.

[0296] A second transfer technique (referred to as bi-directionalcontact transfer) is illustrated in FIG. 6. Here first and secondmembrane stacks 13 a and 13 b sandwich gel slab 54. A pair of filterpads 24 and 26, for instance constructed of a blotting paper such asGB004 Blotter Paper available from Schleicher and Schuell, are providedadjacent to the membrane stacks as shown. Filter pads 24 and 26 aresaturated with a transfer buffer such as TRIS or phosphate base buffers.

[0297] A collapsible, fluid impervious enclosure 28 is provided toenvelop the pads, membrane stacks, and gel as shown in FIG. 6. Plasticbag enclosure 28 is preferably a heat sealable pouch such as thoseavailable from Kapak Corp. (Minneapolis, Minn.). In many embodiments, itis best to remove most of the air from bag 28, for instance by gentlesqueezing and/or vacuum suction. The bag is then sealed by a heat sealersuch as the Impulse Sealer (American International Electric). Enclosure28 is then placed between a pair of heating elements 56 a and 56 b suchas those provided in Gel Dryers manufactured by Bio-Rad Laboratories(Hercules, Calif.). The bag and its contents are preferably heated to atemperature of between about 50 to 90° C., preferably to about 80° C.for about 24 hours. Pressure is preferably applied throughout theheating process using a weight 34. Alternatively, a specific device forapplying heat and/or pressure (such as that illustrated in FIG. 21) canbe employed.

[0298] The heat and pressure applied to contents of the enclosure permitproteins and other molecules to be transferred from the gel to themembrane stack. This produces multiple copies or replicas of thebiomolecular content of the gel.

[0299] In addition to their use in identifying the proteins of theproteome, the methods and kits provided herein can be used to measurethe concentration of a protein (either in absolute terms, or relative tothe concentration of another protein). Likewise, they can be used tomeasure the distribution of variants of a protein, and to identifyproteins that are analogous in structure or function to identified(e.g., human) proteins, or to identify plant clones or transgenicanimals that express a particular protein or protein variant (which maybe linked to, or associated with, a trait or phenotype).

[0300] X. Image Analysis Software

[0301] Software 46 is made available to users of any of the providedkits by providing it on a diskette to be included within the kit (e.g.,kit 36, 58, or 122) or by making it accessible for downloading over theInternet or a private Intranet network, or by other means. The functionof software 46 is to translate the visible spots generated by detectormolecules (such as antibody cocktails 38) into useful information aboutthe proteome of the sample being tested. This information primarilyincludes the quantity of the proteins in the test sample relative to acontrol and, in some cases, information about certain functional aspectsof these proteins. Suitable software can be obtained from, or adaptedfrom, any of a variety of sources (e.g., http://www.2dgels.com/home.htmland http://expasy.proteome.org.au).

[0302] After it is determined which molecules (e.g., proteins) will beidentified on each layer for a given panel/kit, a template image such asthat shown for a 2-D gel (reference numeral 140) is createdcorresponding to each layer (FIG. 25) and stored in software 46. In thisexample, the 2-D gel X-Y coordinates of each protein can be ascertainedfrom any of a number of references and databases. Thus, referring toFIG. 25, template image 140 is the image of what a membrane would looklike if all of the targeted proteins assigned to the layer are presentin the sample being tested along with the landmark “housekeeping”proteins 142 a, 142 b, 142 c. Each antibody cocktail generates a uniquedot pattern on the corresponding membrane to which it is applied as aresult of the selection process outlined above. A template membrane 140will be used by image processing software to analyze experimentalmembranes generated by users. Important feature of the template isexistence of the internal landmarks 142. These spots will originate fromthe set of antibodies targeted against housekeeping proteins present inevery sample regardless of origin. Since their relationship always staythe same these landmarks will serve to normalize samples for loadingdifferences and to compensate for any distortion caused by gel runningprocess.

[0303] Image analysis will start with digitalized image(s) of theexperimental membranes. As the first step, the user matches templateswith the membranes. Software then compares an image of the template andan image of the membrane and performs alignment of spots/bands. The userhas options of visual alignment control and the ability to hand correctminor discrepancies. The second step of analysis will includedensitometric readings of the spots on experimental membranes. This datais stored in the database. The third step includes numerical datamanipulation. Intensity values of each experimental spot on the membraneare divided with values of the landmark spots. This step generatesnormalized intensity values for each spot on the membrane. All the spotsof interest can thus be compared with each other.

[0304] Software 46 preferably allows the user to select the kind ofcomparative analysis to be performed (i.e. comparing the spots or bandspresent in one sample with those in another sample or comparing thosepresent on one membrane with those of another membrane within the samemembrane stack). Results of the analysis are displayed in, for instance,tabular format and user is given the option to go back and comparemagnified sections of the images of interest.

[0305] In one embodiment, software is provided with template imagescorresponding to each of the membrane images. Such software allows theidentity of the protein in each spot to be confirmed based upon thevertical and horizontal position of the protein's spot on the gel.Examples of such software also allow the density of each spot to becalculated so as to provide a quantitative or semi-quantitative read-outas described herein. Such software may also have links to a database ofimages generated from other gels to allow comparisons to be made betweendifferent diseased and normal samples, or links to images or data(structure, sequence, function, etc.).

[0306] In some embodiments, software is also provided to overlay imagesof the bands or spots or cells onto a master image of a ubiquitouslystained sample or gel. A key feature of examples of such software is theability to quantify the biomolecules by determining the density of thebands or spots and comparing them to a control. This process is known as“normalization.” For analysis of 1-D gels a variety of commerciallyavailable programs may be employed such as the 1-D Image AnalysisSoftware available from Eastman Kodak Co.

[0307] Having now generally described the invention, the same will bemore readily understood through reference to the following examples,which are provided by way of illustration, and are not intended to belimiting of the present invention, unless specified.

EXAMPLES Example 1 Construction of Polycarbonate Membranes for ProteinBinding

[0308] Native, non-coated polycarbonate membrane (Millipore, Mass.) haslow affinity and low binding capacity for proteins. To improve itsprotein binding characteristics, polycarbonate membranes were coatedwith either poly-L lysine (referred to as PC+Lysin in FIG. 26) ornitrocellulose (referred to as PC+NC in FIG. 26). Membranes (177 squarecentimeters) were immersed for one minute in 5 ml of either aqueoussolution of 0.1% poly-L-lysine or 0.1-1.0% nitrocellulose solution in100% methanol. After coating, membranes were suspended in verticalposition and air-dried at room temperature for 5-10 minutes.Poly-L-lysine treated membranes were before use additionally baked fortwo hours at 50° C. Small squares (0.25 square centimeters) of bothtreated and non-treated membranes were incubated in TBST solution (50 mMTRIS pH 8.0, 150 mM NaCl and 0.05% Tween-20) with 1.0-100.0 ng/μl ofgoat immunoglobulin labeled with Cy3 fluorescent dye (Amersham PharmaciaBiotech, USA) for 0.5-2 hours at room temperature.

[0309] Membranes were washed in TBST and examined on STORM scanner(Molecular Dynamics, USA). The results are shown in FIG. 26A. Theintensity of the signal was quantified by ImageQuant (MolecularDynamics, USA) and data points from five different experiments wereplotted using Microsoft Excel. The results shown in FIG. 26B demonstratethat polycarbonate membranes have a low protein binding potential thatcan be considerably enhanced by coating the membrane with poly-L-lysine(PC+Lysin) or nitrocellulose (PCNC).

Example 2 Testing the Porosity of Prepared Polycarbonate Membrane Layers

[0310] To demonstrate porosity of manufactured layers, native,poly-L-lysine or nitrocellulose coated membranes were blocked in 5%bovine serum albumen solution in 50 mM TRIS pH 8.0 to prevent anyprotein binding. Fifty-one square centimeter pieces were cut out andstacked together to make a pile. A non-blocked pure nitrocellulose layerwas used at the bottom to capture proteins (NC-trap). Three adjacent 20micrometer thick frozen sections of normal breast tissue were collectedon a polycarbonate membrane with 5.0 um pore size and embedded in a 2%agarose gel and transferred side by side through each stack. Between 50and 100 milliliters of TBST buffer was used per square centimeter of themembrane stack with average length of the transfer being 1 hour. Aftertransfer, proteins remaining in the tissue sections and total proteinson the NC-trap were visualized by Ponceau S staining (SIGMA, Mo.).

[0311] As shown in FIG. 27, the outline of the total proteinstransferred through the stack and trapped on the nitrocellulose layervery closely resembled the outline of the applied tissue section. Thissuggests that not only were membranes porous enough to allow for theproteins to be transferred, but also that at least up to 50polycarbonate membranes can be used in this kind of assay withoutapparent distortion of the image due to lateral diffusion.

EXAMPLE 3 Demonstration of Low Capacity Protein Binding toNitrocellulose Coated Polycarbonate Layers

[0312] Examples 1 and 2 demonstrate that proteins in solution can bindto a single nitrocellulose coated polycarbonate layer and that completesaturation of the layer with proteins does not affect its porosity. Toascertain how much of the total protein would be trapped on eachindividual layer during the tissue section transfer, 20 micron thickfrozen sections of normal and tumor breast tissue were placed adjacentto each other on a polycarbonate membrane with 5.0 um pore size,embedded in 2% agarose gel and transferred through 14 layers ofnitrocellulose coated polycarbonate to the NC-trap on the bottom, in 100ml/cm² of buffer containing 25 mM TRIS pH 8.3, 192 mM glycine, 0.05% SDSand 20% methanol. After transfer, proteins left over in the tissuesections were visualized by Ponceau S staining (SIGMA, U.S.A.) and totaleluted proteins captured on the NC-trap were visualized by BLOTFastStain (Chemicon, USA). The image formed on the trap demonstratedsuccessful transfer of the protein through the membranes.

[0313] To determine whether sufficient total protein trapped on eachmembrane during the transfer to perform immunological detection 14arbitrarily selected antibodies were used. Antibodies were: Anti-GAPDH,1:100 (Chemicon, MAB374); Anti-Rsk, 1:1,000 (Transduction Laboratories,R23820); Anti-Stat5a, 1:500 (Santa Cruz Biotechnology, sc-1081);Anti-IFNalpha, 1:500 (Biosource, AHC4814); Anti-RARalpha, 1:1,000(Biomol, sa-178); Anti-phospho-EGFR, 1:1,000 (Upstate, 05-483);Anti-non-phospho EGFR, 1:1,000 (Upstate, 05-484); Anti-phospho-NR1,1:500 (Upstate, 06-651); Anti-Stat1, 1:2,000 (Transduction Laboratories,G16920); Anti-Rb, 1:1,000 (Santa Cruz Biotechnology, sc-50); Anti-Jak1,1:500 (Santa Cruz Biotechnology, sc-295); Anti-tubulin-alpha, 1:500(Santa Cruz Biotechnology, sc-5546); Anti-beta-actin, 1:2,000 (SIGMA,A5441).

[0314] Polycarbonate layers were first blocked in 1× casein solution(Vector Labs, U.S.A.) for one hour at room temperature and incubatedovernight at 4° C. in primary antibodies as listed in FIG. 28 followedby TBST washes and incubation in alkaline phosphatase (AP) conjugatedsecondary antibodies (1:2,000 dilution) (Rockland, U.S.A.). Membraneswere then incubated for five minutes in enhanced chemiluminescence (ECL)substrate (Vector Labs, USA) followed by visualization of the protein byexposing the membranes to X-ray film (Kodak, USA).

[0315] The results showed that methods provided herein allow detectionof a number of different proteins. To ascertain how the membranesperformed with respect to the amount of total protein captured, themembranes were each incubated with the same antibody, allowingdetermination of the protein content on each of them. Anti-GAPDHantibody was used for three hours at room temperature, washed in TBST,incubated with anti-mouse secondary antibody conjugated to horseradishperoxidase (HRP) and visualized in enhanced chemiluminescence substratespecific only for HRP (Pierce, USA). After ECL reaction membranes wereexposed to film as stated before. The results confirmed that all of themembranes did capture a similar portion of the total protein anddifferences seen in the first part of the experiment are not the resultof differences in membrane “loading.” For documentation purposes, theX-ray film was scanned on the flat bed scanner (Lacie, USA) and imageswere processed using ADOBE PhotoShop 4.0.

Example 4 Transferring Proteins from Tissue Microarrays

[0316] A five microns (5 μM) thick paraffin section of a tissuemicroarray originating from the National Institutes of Health (NIH)Tissue Array Research Program (TARP) was collected on tape andtransferred through four membranes in the manner described above. Themembranes were as provided above. The transfer solution contained 50 mMTRIS and 380 mM glycine. This yields a buffer with approximately pH 8.6,but this can be adjusted to anywhere in a range of pH 8.0 to 9.5.

[0317] Plastic pouch enclosing membrane stack and tissue array wasplaced in a Gel Drier (BioRad) and lid of the drier was used as apressure and heating (80° C.) source. Heat and pressure were applied fortwo (2) hours.

[0318] After transfer, membranes were gently washed in TBST buffer (50mM TRIS pH 8.0, 150 mM NaCl and 0.05% Tween-20) and stained with FASTBlue Stain (Chemicon) according to manufacturer instructions. Scanningon an Astra 2200 scanner (UMAX) digitalized images of the individuallayers. After staining, membranes were rinsed in TBST buffer, blockedfor 15 minutes in 1× casein solution (Vector Laboratories, Inc.) andincubated overnight at 4° C. in primary antibody (anti-cytokeratin(1:5,000, Sigma) or anti-PSA (1:500, Script)). The membranes where thenwashed in TBST, incubated in the complex of secondary antibody andalkaline phosphatase, and washed again. Localization of the targetprotein (tytokeratin or PSA) was visualized by enhancedchemiluminescence (ECL) (DuoLux, Vector Laboratories, Inc.) and BiomaxMR film (Kodak). The images were digitalized by scanning on an Astra2200 scanner (UMAX).

[0319] The results, shown in FIG. 29, demonstrate that membrane replicascan be made from tissue arrays by using the described techniques withoutloosing spatial resolution. It also demonstrate that immunodetection ofa single protein is possible on these membranes.

Example 5 Differential Protein Expression in Different Tumors

[0320] Membrane copies of the TARP array were prepared and assayed asstated in the previous example. For detection, the following primaryantibodies were used: anti-cytokeratin (1:5,000, Sigma), anti-PSA(1:500, Script), anti-p53 (1:1,000, Transduction Laboratories) andanti-p300 (1:500, Transduction Laboratories).

[0321] The results, shown in FIG. 30, demonstrate that different tumortypes express different amounts of the same protein (for instance, PSAis primarily expressed in the prostate cancer samples) and that the sametumor type can express different amount of the same protein (forinstance, p53 and p300 are expressed in only a subset of colon carcinomasamples).

Example 6

[0322] Immunodetection on Membranes Using Antibodies Ineffective in IHIC

[0323] Membrane copies obtained from the transfer of normal humantonsilar tissue, normal human kidney tissue, and TARP tissue array wereproduced as described in the previous examples. In some cases, themembranes were subjected to antigen retrieval, by immersing them in asolution of 0.1 M sodium citrate containing 10 mM EDTA pH 8, for 5minutes, at 95° C.

[0324] Following blocking in a 1× casein solution (Vector Laboratories)for 30 minutes, the membranes were incubated with monoclonal antibodiesdiluted at 1:20-1:50 for 16 hours at 4° C. Primary antibodies were usedessentially as directed in the manufactures' instructions; each of theantibodies selected are noted by the manufacture to be ineffective whenused to detect proteins in formalin preserved tissue samples, even whenthe samples are subjected to antigen retrieval. The following antibodieswere used: anti-CD3 (CALTAG); anti-EGFR; anti-Progesterone Receptor;(Dako); and anti-erbB2 (Zymed). Following TBST washes, proteins werevisualized as described in Example 4.

[0325] In each case, the antibody yielded clear signals on thetransferred membranes but would not yield signals when used for mHC onadjacent sections, directly on a corresponding microarray. Thus,transfer of biomolecules to membranes using the described contacttransfer method is effective for immunodetection visualization usingantibodies that are ineffective in IHC.

Example 7 Transferring Proteins from Cells Collected by LCM to Membranes

[0326] Five microns thick frozen section of squamous carcinoma of thehead and neck was collected on plain glass slide. The slide was fixed in100% ethanol for three minutes, immersed in 0.5% ethanol solution ofAzure Blue (SIGMA) for one minute followed by five minutes incubation inxylene. LCM was performed as recommended by the manufacturer (Arcturus).Each LCM cap received approximately 50 laser hits, corresponding to250-300 cells during a 15-20 minute time period. Immediately after this,caps were stored at −80° C. until transfer.

[0327] Just prior to transfer, caps were hydrated through an ethanolgradient and transfer was assembled as shown in FIG. 8. Five differentmembrane layers were used. Transfer buffer contained 25 mM TRIS, 192 mMglycine and 0.025% SDS. The assembled package was placed in a gel drier(BioRad) and lid of the drier was used as a pressure and temperature(80° C.) source. The transfer process took about two hours.

[0328] After transfer, the stack was disassembled, membranes were washedin TBST buffer (50 mM TRIS pH 8.0, 150 mM NaCl and 0.05% Tween-20) andthen stained with FAST Blue Stain (Chemicon) according to manufacturerinstructions. Scanning on Astra 2200 scanner (UMAX) produced digitalisedimages of the layers. After staining, membranes were rinsed in TBSTbuffer, blocked for 15 minutes in 1× casein solution (VectorLaboratories, Inc.) and incubated overnight at +4° C. inanti-cytokeratin antibody (1:5,000, Sigma), washed in TBST, incubated inthe complex of secondary antibody and alkaline phosphatase, washed againand location of the protein was visualized by ECL (DuoLux, VectorLaboratories, Inc.) and Biomax MR film (Kodak). The resultant image wasdigitalised by scanning on an Astra 2200 scanner (UMAX). FIG. 31 shows“copies” that were made on five membranes, and that antibodies wereeffectively used to detect proteins on each layer.

Example 8 Transfer and Capture of Proteins From a 1-D Gel

[0329] This example demonstrates that polycarbonate coatednitrocellulose (PCNC) membranes, with their high binding affinity butlow capacity for the proteins eluted from the gel, can be used to makemultiple copies of a gel.

[0330] 1.0 μg/lane of biotinylated protein marker (Vector Laboratories,Inc) was separated by 15% PAGE and electro-transferred in 25 mM Tris,192 mM glycine, 0.025% SDS and 20% methanol (60-110 V for 1-2 hours)through a stack of PCNC membranes; the number of membranes per stack was5-20, depending on the experiment At the end of the stack, one purenitrocellulose membrane was used to capture proteins that were not boundto PCNC layers (“NC trap”). Transfer was performed from 0.5-3 hours at60-110 V in a Ready Gel Cell apparatus (BioRad).

[0331] After transfer, membranes were rinsed in 50 mM Tris pH 8.0 and150 mM NaCl (TBS buffer), blocked for 10-60 minutes in 1× caseinsolution (Vector Laboratories, Inc.), and incubated for 30 minutes inVECTASTAIN ABC-AmP reagent (Vector Laboratories, Inc.). Membranes werewashed again in TBST, rinsed in 0.1 M TRIS pH 9.5, incubated in DuoLuxreagent (Vector Laboratories, Inc.) for 3-5 minutes, and exposed toBiomax MR film (Kodak). An example of one representative experiment isshown in FIG. 32.

[0332] Results demonstrated that:

[0333] 1. PCNC stack of membranes did not interfere with post-transferWestern blotting procedure—proteins were transferred from the gelthrough the stack and to the NC trap;

[0334] 2. A wide range of protein sizes were transferred through thestack with very similar transfer efficiency—7 kDa-200 kDa proteins weredetected on the NC trap; and

[0335] 3. PCNC layers captured proteins regardless of their size.

[0336] In order to determine compatibility of PCNC membranes withimmunodetection, Jurkat cell were lysed in buffer (50 mM TRIS pH 8.0 and1% SDS) and 20 μg/lane of total protein was separated by 15% PAGE. Theresultant gel was electro-transferred through a stack of PCNC membranesin 25 mM TRIS, 192 mM glycine, 0.025% SDS and 20% methanol. Transfer wascarried out at 60-110 V for one to two hours.

[0337] All of the membranes were incubated in primary anti-Rsk (1:100,Transduction Laboratories) and anti-p300 (1:500, TransductionLaboratories) antibody, washed in TBST buffer, incubated with thecomplex of secondary antibody and alkaline phosphatase, and washedagain. The location of the protein on the blots was visualized using ECL(DuoLux, Vector Laboratories, Inc.) and Biomax MR film (Kodak). Theresults, shown in FIG. 33, demonstrated that PCNC membranes are suitablefor this type of protein detection. Each membrane captured sufficientprotein to be detected by immunological methods, but each singlemembrane did not capture too much protein, enabling a number of copiesof the same gel to be generated.

Example 9 Transfer and Capture of Proteins From a 2-D Gel

[0338] 2-D protein gels have greater separation capabilities than 1-Dgels. Two-dimensional separation allows identification of hundreds oreven thousands of proteins on the same gel. Proteins separated by 2-Dgels are identified by protein sequencing or immunological features.Sequencing requires expensive equipment, highly trained operators, andits use is limited to a small number of privileged groups.Immunodetection is easier to do but it is a low throughput technique,since traditional blotting procedures generate only one membrane pergel.

[0339] As described above, one can make 10 or more 1-D gel copies usingPCNC membranes. In order to find out if 2-D gels can be “copied” thesame way, the proteins present in 500 μg of Jurkat cell protein lysatewere separated by 2-D PAGE. A commercial immobilized pH gradient (IPG)from 3.0 to 10.0 (Pharmacia Biotech, Uppsala, Sweden) was used forfirst-dimension separation. Eight to 12 hours of in-gel samplerehydration was used for protein loading. Proteins were separated for atotal of 15,000-30,000 Vhrs. After equilibration, the IPG strips weretransferred onto vertical gradient gel (4-20%, Novex) for seconddimension separation.

[0340] After electrophoresis, the 2-D gel was transferred in 25 mM Tris,192 mM glycine, 0.025% SDS and 20% methanol (60-110 V for 1-2 hours)through a stack of five PCNC membranes. The membranes were then rinsedin TBST buffer, then blocked for 10-60 minutes in 1× casein solution(Vector Laboratories, Inc.) prior to probing with specific antibodies.Individual membranes were probed by incubating them overnight at 4° C.in anti-GAPDH (1:5,000, Chemicon), anti-beta-actin (1:5,000, Sigma)and/or anti alpha-tubulin (1:1,000, Calbiochem) antibody. The membraneswere then washed in TBST, incubated in the complex of secondary antibodyand alkaline phosphatase, and washed again. The location of the proteinwas visualized by ECL (DuoLux, Vector Laboratories, Inc.) and Biomax MRfilm (Kodak).

[0341] Antibodies were first applied separately to three differentmembranes (from three different gels) to find the precise spatiallocation of specific proteins in the 2-D gel. These three proteins(GAPDH, actin, and tubulin) differ in their size and charge, and werespatially separated from each other on the gel.

[0342] In order to increase the throughput of immuno-detection, allthree antibodies were mixed together and applied as a detector cocktailto all five membranes from the same gel. The results of this experimentare shown in FIG. 34. Generating multiple replicas of the same gel andusing an antibody cocktail approach increased throughput of theimmunological protein identification on 2-D gels.

Example 10 Use of Layered Membranes for Protein-DNA ComplexesIdentification

[0343] The following example demonstrates the ability of the layeredmembranes of the present invention to speed up and simplify theidentification of the proteins of a protein-DNA complex. It shows thatcopies of the gel were made and each of the membranes was successfullyimmuno-probed with a different antibody of interest.

[0344] 250 ng of recombinant his6-c-rel and 120 ng of purifiedrecombinant his6-CREB were incubated alone or in combination with 0.2 ngof ³²P-5′ labeled duplex oligonucleotide (SEQ ID NO: 1), in 10 μl ofbuffer containing 10 mM HEPES, 50 mM NaCl, 20% glycerol, and 4 mM PME.The hybridization reaction was allowed to proceed at room temperaturefor 30 minutes. Samples were separated by electrophoresis on 4%polyacrylamide gel at 180 Volts for one hour, then transferred in 25 mMTRIS, 192 mM glycine, 0.025% SDS and 20% methanol (60-110 V for 1-2hours) through a stack of four PCNC membranes (as described herein) andone NA45 DEAE membrane (Schleicher & Schuell). This last layer ofcharged cellulose was used to trap DNA released from the gel thattransferred through the entire thickness of the stack. After transfer,registration (orientation) marks were made using a 19G needle. The DEAEmembrane was dried, exposed overnight to a phosphoimager screen, andvisualized on a Phosphorimager: SI (Molecular Dynamics).

[0345] First and second PCNC membranes were rinsed in TBST buffer,blocked for 10-60 minutes in 1× casein solution (Vector Laboratories,Inc.) and incubated overnight at 4° C. in anti-rat antibody (1:200, NCILaboratory of Pathology, Transcription Regulation Unit Chief, Dr. KevinGardner) and anti-His (1:10,000, Stratagene). The membranes were washedin TBST, incubated in the complex of secondary antibody and alkalinephosphatase, then washed again. The location of the specific proteinswas visualized by ECL (DuoLux, Vector Laboratories, Inc.) and Biomax MRfilm (Kodak). Images of all of the membranes were aligned in AdobePhotoshop (FIG. 35).

[0346] The results demonstrated that the layered membrane array providesfast and reliable identification of proteins from a protein complex.

Example 11 Uniformity of Protein Capture on Multiple Membranes

[0347] During electrotransfer, proteins are pushed (or pulled) out ofthe gel onto the membrane substrate. The speed of their migration isinfluenced by the magnitude of the electric current and size of theprotein. A higher voltage will push proteins out of the gel faster thena lower voltage. Even with fixed current flow, smaller proteinsgenerally move faster then larger ones. The length of the transfer isanother variable that can influence quality of membrane copies. Iftransfer is too short, not enough of the protein will leave the gel andbe accessible for binding onto the membranes.

[0348] An analysis of the results obtained with the methods andmaterials described herein indicates that, regardless of the amount ofprotein that is present in the gel, more uniform membrane copies can begenerated if transfer is performed for shorter time with higher voltage.All of the transfers in this Example are performed for 60-70 minutes at59-63 volts. Keeping the transfer conditions constant, the influence ofprotein load amount on the ability to create membrane copies wasexamined.

[0349] Total protein extracted from the Jurkat cell line (cells oflymphatic origin), the HN12 cell line (epithelial cells of keratinocyteorigin) and the SW480 cell line (cells of adeno-epithelial origin) wereused for this Example. All cell lines were cultured in a 37° C.humidified incubator in DMEM media with 10% added serum. At about 80-90%confluence, cells were harvested by scraping them from the dish; thecells were then resuspended in phosphate buffered saline (PBS) with 1%added SDS. The concentration of the total protein was determined by BCAProtein Assay Reagent (Pierce). Approximately 30-100 micrograms of totalprotein was separated by 4-20% polyacrylamide gel electrophoresis (PAGE)(BioRad). A suitable protein gel running buffer was used in theelectrophoresis to separate the proteins (for instance, 25 mM TRIS pH8.3, 192 mM glycine, 0.1% SDS). In addition, protein size markers(Bio-Rad Kaleidoscopic Standard, catalog number 161-0324) were loaded onthe gel.

[0350] After electrophoretic separation, proteins were transferredthrough a 10-layered array by electroblotting (Bio-Rad catalog number170-3930). A fiber pad, or more than one fiber pad was used at the anodeand the cathode during electroblotting. Thus, starting from the cathodeside of the electroblotting cassette, the fiber pad (on the bottom ofthe sandwich), filter paper, gel, and membrane stack are layered inorder, with one membrane (the first membrane, denoted membrane “1”) incontact with the gel. When assembled, the electroblotting cassettetightly squeezes the “transfer sandwich” (unlike a single membranetransfer, which can be gently squeezed). Fiber pads may be added on theoutside of the sandwich umtil the cassette seems “overfilled.” When thesandwich has the proper thickness, it may be necessary to force thecassette closed.

[0351] The electroblotting procedure will vary depending on the systemused (for Bio-Rad devices, transfer is accomplished at 59-63 volts for60-70 minutes; for Novex devices, transfer is accomplished at 25 voltsfor 120 minutes). To facilitate later labeling of individual membranes,holes can be punched (for instance, using a 23 g-25 g needle)distinguishable locations, such as in the center of each proteinstandard band, and in the center of each well.

[0352] After transfer, the membrane stack was removed from the gel bygently peeling up one corner, and the frames were opened or removed. Themembrane stack was then washed in Tris or phosphate buffered solution,and the membranes separated while they are still in the solution. Beforeimmunodetection, the membranes are immersed in Blocking Reagent (20/20GeneSystems) for 15 minutes.

[0353] Membranes were separately stained with Sypro Ruby (MolecularProbes) as recommended by the manufacturer and visualized on an ImageStation 440CF (Kodak). Fluorescence intensities were taken from threedifferent regions of every sample on every membrane using KODAK ID ImageAnalysis Software (Kodak). The first region included proteins from 20-40kDa in size. The second region included proteins from 40-100 kDa insize. The third region included proteins 100-150 kDa in size. Therelationship between different groups was analyzed using MicrosoftExcel®.

[0354] A data plot (FIG. 36) demonstrates that the smallest variabilityin total protein loading per membrane was seen for proteins 40-100 kDain size. The data also suggest that the amount of protein loaded was animportant variable in this system. For proteins that are 40-100 kDa insize, it was determined that loading of 70-100 μg per lane keptvariability between the membranes in the less than 10% range.

Example 12 Detecting Presence and Functional State of Multiple ProteinsSeparated on a Single Gel

[0355] To determine the feasibility of detecting the presence andfunctional state of multiple proteins from the same gel, the presenceand functional state of EGFR and c-myc protein was checked in parallel.Samples used were from the Jurkat cell line, HN12 cell line, and SW480cell line; cells were cultured and harvested as stated in Example 11.Thirty micrograms of total protein was loaded per lane of 4-20%polyacrylamide gel (BioRad) and separated for two hours at 50 V. Afterelectrophoresis, the gel was equilibrated for 10 minutes in 1× transferbuffer from 20/20 Gene Systems, Inc. and electrotransfer was assembledwith a seven-layered membrane stack (20/20 Gene Systems, Inc). AMiniCell blotter (BioRad) was used for the electrotransfer. Transfer wasperformed for 60-70 minutes at 59-63 V. After transfer, membranes wereseparated in 50 mM Tris pH 8.0, 150 mM NaCl and 0.05% Tween-20 (TBST),blocked in 1× casein solution (Vector) for 15 minutes at roomtemperature and incubated with antibodies diluted in TBST as indicatedin TABLE 1 for 12 hours at 4° C. TABLE 1 Layer Number ProteinManufacturer Part Number Ab Dilution 1 Total EGFR Neomarker MS-610 1:5002 Total EGFR Santa Cruz SC-03 1:200 3 Phospho-EGFR Upstate 05-4841:1,000 4 Phospho-EGFR Upstate 05-483 1:1,000 5 Total c-myc Santa CruzSC-764 1:200 6 Total c-myc Neomarkers MS-127 1:500 7 Phospho-myc CellSignaling 9401L 1:1,000 1-7 Alpha-tubulin Calbiochem CP06 1:500

[0356] After incubation with primary antibody, each membrane was washedseparately three times for five minutes each in TBST and incubated in1:4,000 dilution of horseradish peroxidase (HRP) conjugated secondaryantibody (Amersham) in Ix casein solution for 30 minutes at roomtemperature. Membranes were then washed for five minutes in TBST andtwice for five minutes each in 50 mM TRIS pH 8.0, 150 mM NaCl (TBS),incubated for five minutes in ECL PLUS substrate (Amersham) and exposedto Biomax MR film (Kodak) from 1-45 minutes.

[0357] The image of the film was digitized on an Astra 2200 scanner(Umax) and manipulated in ADOBE Photoshop 5.0. Following incubation inthe first set of antibodies, all of the membranes were incubated inanti-alpha-tubulin antibody for two hours at room temperature and signalvisualized as stated above, with the exception that the secondaryantibody was conjugated with alkaline phosphatase (AP) (Vector) and theECL reagent used was DuoLux (Vector).

[0358] The result of this Example, shown in FIG. 37, demonstrated thatmultiple membrane copies made from the same gel could be used todetermine the presence and functional state of multiple proteins fromthe same sample: In this Example, both total and activated forms of EGFRand c-myc protein were detected in extracts prepared from the SW480 cellline. Results also demonstrated that different samples could be comparedto each other to reveal the presence of total protein (for instance,EGFR was expressed in HN12 and SW480 cells, but not in Jurkat cells) andthat the presence of total protein does not necessarily mean functionalactivity (c-myc was present in both Jurkat and SW480 cell lines but onlyJurkat cells had an active, functional form).

Example 13 Detecting Proteins Involved in Epidermal Growth FactorReceptor (EGFR) Signaling Pathway

[0359] Advantages of certain of the encompassed embodiments include thatthey permit analysis and comparison, in parallel, of a number ofdifferent proteins from multiple samples. The value of this parallelapproach is even greater where the proteins of interest belong to asingle biological system (e.g., all are component s of a receptorsignaling pathway). Since analysis for all of the proteins is done on asingle sample, comparative studies are easier to perform, and it isexpected that the results are more consistent and reliable.

[0360] In this Example, the functional state of nine proteins that areinvolved in signaling through the EGFR pathway were analyzed andcompared. Four different keratinocyte cell lines were cultured andharvested as described above (see Example 11). One hundred micrograms oftotal protein from each cell line was separated on a 420% acrylamidegradient gel (BioRad) and transferred through a ten-layered array asdescribed above (see Example 11).

[0361] Membranes were stained with the ubiquitous dye Sypro Ruby(Molecular Probes) and images captured and stored on Image Station 440CF(Kodak). Following visualization of the total protein, membranes wereblocked in 1× casein solution (Vector) for 15 minutes at roomtemperature, then incubated with antibodies diluted in TBST as indicatedin TABLE 2 for 12 hours at 4° C. The control membrane layer wasincubated in no primary antibody. TABLE 2 Protein Manufacturer PartNumber Ab Dilution Detected Phospho-Raf Biosource 44-504 1:1,500 YesPhosho-Akt Cell Signaling 9276S 1:1,000 Yes Phosho-Erk Cell Signaling9106S 1:1,000 Yes Phosho-Myc Cell Signaling 9401L 1:1,000 YesPhosho-EGFR Upstate 05-493 1:1,000 Yes Total EGFR Neomarkers MS610 1:500Yes Phospho-Stat3 Biosource 44-384 1:1,000 Yes Phospho-PKC CellSignaling 2261 1:1,000 No Phospho-Src Biosource 44-660 1:1,000 Yes

[0362] Following incubation with primary antibodies, membranes wereprocessed as described above (see EXAMPLE 12).

[0363]FIG. 38 shows that eight of the nine proteins tested could bedetected on the stacked membranes using this method. The phosphorylatedform of PKC was not detected in these samples. Follow up experimentsalso failed to detect this form of protein when the same amount ofsample was blotted on single nitrocellulose membrane with positivecontrol cellular extract being positive (PDGF treated 3T3 cells, 10μg/lane, provided by Cell Signaling). This suggests that the failure todetect the phosphorylated form of PKC was not due to a deficiency in thetransfer system but to the very small (if any) amount of this proteinpresent in the tested cell lines. The results also clearly illustratedifferential expression between different cell lines for all of theproteins tested.

Example 14 Contact Transfer of Proteins From a 1-D Gel

[0364] Diffusion based transfer of proteins from an acrylamide gel ontosingle membrane substrate was previously discussed by Bowen et al(Nucleic Acid Res., 8:1-20, 1980). The apparent advantage of this systemis that it does not require special blotting equipment. This Example wascarried out in order to determine if it is possible to use contacttransfer (without applying an electric current) with the providedmembrane arrays.

[0365] A 10% gel (BioRad) with 25 and 50 micrograms of total protein wassandwiched between two five-membrane membrane stacks as shown in FIG. 6,with five membranes on each side of the gel. Three layers of Whatman®filter paper soaked in 1× transfer buffer from 20/20 Gene Systems, Inc.were added on each side of the sandwich and the whole, assembled stackwas sealed in a plastic bag. Three parallel sample stacks were assembledand placed in a gel drier (BioRad) with the lid closed at 80° C.Individual sample stacks were removed after 30, 60 or 120 minutes oftransfer. After transfer, the membranes were washed in TBST and stainedwith FastBlue Stain (Calbiochem) as recommended by the manufacturer.Stained membranes were scanned using an Astra 2200 scanner (Umax) todetect total transferred protein, and the images were manipulated inADOBE Photoshop 5.0.

[0366] The results of this procedure are shown in FIG. 39. Proteins wereeffectively transferred from the gel into the membranes on both sides ofthe gel (bi-directional transfer). The amount of the protein transferredwas dependent on the length of the transfer (more protein wastransferred after two hours compared to half an hour) and the size ofthe protein (transfer of the large proteins was less efficient). Thus,contact transfer is an effective alternative to electrotransfer ofproteins and other biomolecules onto/into membrane stacks.

[0367] Although certain embodiments have been described herein, it willbe apparent to those skilled in the art to which the invention pertainsthat variations and modifications of the described embodiments may bemade without departing from the spirit and scope of the disclosure.Accordingly, it is intended that the invention be limited only to theextent required by the appended claims and the applicable rules of law.The references cited above are incorporated herein in their entirety.

1 1 1 43 DNA Artificial sequence Synthetic oligonucleotide 1 tcgacctcttctgatgactc tttggaattt ctttaaaccc cca 43

1. A method of detecting biomolecules in a sample comprising: providinga stack of at least two layered membranes; applying the sample to thestack under conditions that permit movement of the biomolecules throughmultiple layered membranes of the stack, and allow direct capture of atleast a portion of the biomolecules on the membranes; and detecting thebiomolecules on one or more of the multiple membranes.
 2. The methodaccording to claim 1 wherein the stack comprises a plurality of poroussubstrates each having a thickness of less than 30 microns.
 3. Themethod according to claim 2 wherein one or more of the substratescomprise a material for increasing the affinity of the membrane to thebiomolecules.
 4. The method of claim 3, wherein the material is coatedon one or more of the membranes.
 5. The method of claim 2 wherein theporous substrates comprise a material selected from the group consistingof polycarbonate, cellulose acetate, and mixtures thereof.
 6. The methodof claim 5, wherein the porous substrate comprises a polycarbonatesubstrate.
 7. The method of claim 5, wherein the material for increasingaffinity is selected from the group consisting of nitrocellulose,poly-L-lysine, and mixtures thereof.
 8. The method of claim 5, whereinthe material for increasing affinity is a biomolecule-specific ligand.9. The method of claim 5, wherein the porous substrate comprises apolycarbonate substrate and the material for increasing affinitycomprises nitrocellulose.
 10. The method according to claim 1 whereinthe sample is a tissue section.
 11. The method of claim 1, whereindetecting the biomolecules comprises separating one or more of themembranes from the stack, and detecting the biomolecules on the one ormore of the separated membranes.
 12. The method of claim 1, wherein theconditions that permit movement of the biomolecules through the multiplemembranes comprises passing a transfer liquid through the layeredmembranes.
 13. The method of claim 1, wherein the conditions that permitmovement of the biomolecules through one or more of the membranescomprises providing a wick that encourages movement of the biomoleculesthrough the stack of layered membranes in a desired direction ofmovement.
 14. The method of claim 1, wherein the stack of layeredmembranes comprises 5 or more membranes.
 15. The method of claim 14,wherein the stack of layered membranes comprises 20 or more membranes.16. The method of claim 14, wherein the stack of layered membranescomprises 50 or more membranes.
 17. The method of claim 1, wherein thesample comprises a nucleic acid, a protein, a lipid, a carbohydrate, ora combination or mixture thereof.
 18. The method of claim 1, wherein thesample is a substantially two-dimensional sample.
 19. The method ofclaim 18, wherein the substantially two-dimensional sample is selectedfrom the group consisting of a tissue section, a tissue microarray, aLCM harvested sample, a “one-dimensional” electrophoretic gel, a“two-dimensional” electrophoretic gel, a structurally transformedsample, or a combination of two or more thereof.
 20. The method of claim1, further comprising correlating the biomolecules detected on the oneor more membranes with a biological characteristic of the sample.
 21. Amethod of making multiple substantial copies of a biological sample,comprising: providing a stack of layered membranes, wherein themembranes permit biomolecules applied to the stack to move through aplurality of the membranes, while directly capturing at least a portionof the biomolecules on multiple membranes; and applying the biologicalsample to the stack, under conditions that allow the multiple membranesto directly capture the biomolecules from the sample and form themultiple substantial copies of the biological sample, thereby makingmultiple substantial copies of the biological sample.
 22. The method ofclaim 21, wherein the biological sample comprises a nucleic acid, aprotein, a lipid, a carbohydrate, or a combination or mixture thereof.23. The method of claim 21, wherein the biological sample is selectedfrom the group consisting of a tissue section, a tissue microarray, aLCM harvested sample, a “one-dimensional” electrophoretic gel, a“two-dimensional” electrophoretic gel, a structurally transformedsample, or a combination of two or more thereof.
 24. The method of claim21, further comprising detecting one or more biomolecules of interest onat least one of the multiple substantial copies.
 24. The method of claim24, wherein detecting biomolecules of interest comprises exposing aplurality of the multiple membranes to at least one detector molecule.25. The method of claim 24, wherein the biological sample is a tissuespecimen that is placed on the stack of layered membranes, andbiomolecules from the tissue specimen are directly captured by thelayered membranes as the biomolecules from the tissue specimen movethrough the multiple membranes.
 26. The method of claim 24, furthercomprising separating the multiple membranes prior to detecting thebiomolecules of interest.
 27. The method of claim 24, wherein thebiomolecules applied to the stack themselves comprise detectors that areexposed to a biological specimen to be analyzed, and the method furthercomprises exposing one or more of the multiple membranes to thebiological specimen under conditions that allow the biological specimento be analyzed by the detectors.
 28. The method of claim 27, wherein atleast one biomolecule of interest on the multiple membranes is a nucleicacid molecule, and detecting biomolecules of interest comprises exposingthe nucleic acid molecules on the multiple membranes to the biologicalspecimen to be analyzed, under conditions that allow hybridizationbetween the nucleic acid molecules on the membranes and nucleic acidmolecules in the biological specimen.
 29. A method of creating a set ofmicroarray copies comprising: providing a stack of layered membranes;and applying a plurality of DNA probes, antibodies, or a combinationthereof, to the stack of layered membranes, wherein the stack of layeredmembranes comprises a plurality of substrates through which the probesor antibodies move, and in which a portion of the probes or antibodiesare directly captured by one or more of the substrates.
 30. The methodof claim 29, further comprising separating the substrates to providecorresponding substrates having a plurality of the DNA probes,antibodies or combination thereof, in corresponding positions of each ofthe substrates.
 31. The method of claim 29, wherein applying theplurality of DNA probes, antibodies, or combination thereof, is appliedto the stack from a plate having a plurality of wells each containing adifferent DNA probe or antibody, and the DNA probes or antibodies aretransferred from the wells to the stack so as to create a set ofsubstantially replicate microarrays.
 32. A method of analyzingbiomolecules in a tissue sample, comprising: providing at least onemembrane; positioning the at least one membrane in contact with thetissue sample; applying heat and/or pressure to the tissue sample,whereupon biomolecules are transferred from the tissue sample onto theat least one membrane; and, analyzing the biomolecules on the at leastone membrane.
 33. The method of claim 32, wherein the tissue sample isan archival tissue sample, a cryo-preserved tissues ample, a freshtissue sample, an LCM-harvested tissue sample, or a tissue microarray.34. The method of claim 32, comprising providing a plurality ofmembranes and further comprising analyzing the biomolecules on two ormore of the plurality of membranes.
 35. The method of claim 32, whereinthe membrane is a porous membrane of no more than 30 microns thickness,comprising a core substrate and a coating.
 36. The method of claim 35,wherein the core substrate comprises polycarbonate.
 37. The method ofclaim 35, wherein the coating comprises nitrocellulose.
 38. A method ofreplicating biomolecular content of a tissue microarray, comprising:providing the tissue microarray; and transferring biomolecules from thetissue microarray onto a plurality of membranes so as to produce atleast one replicate of the biomolecular content of the tissuemicroarray.
 39. The method of claim 38, wherein transferringbiomolecules comprises: positioning the plurality of membrane in contactwith the tissue microarray; and applying heat and/or pressure to thetissue microarray, whereupon biomolecules are transferred from thetissue microarray onto at least one membrane of the plurality ofmembranes.
 40. A method of analyzing cellular material embedded on anLCM transfer film comprising: providing one or more membranes;positioning the one or more membranes adjacent to the LCM transfer film;transferring biomolecules from the cellular material to the one or moremembranes; and detecting the biomolecules on the membranes.
 41. Themethod of claim 40, wherein transferring biomolecules comprises:applying heat and/or pressure to the membranes and/or the LCM transferfilm, whereupon biomolecules are transferred from the LCM transfer ontothe one or more membranes.
 42. A method for analyzing the proteome of abiological sample comprising: separating at least one protein fromanother protein present in the biological sample; transferring a portionof the separated protein to a plurality of membranes in a stackedconfiguration; incubating each of the membranes in the presence of oneor more species of predetermined ligand molecules under conditionssufficient to permit binding between the separated protein and a ligandcapable of binding to such protein; and analyzing the proteome bydetermining the occurrence of binding between the protein and any of thespecies of predetermined ligand molecules.
 43. The method of claim 42,wherein separating the at least one protein from another protein presentin the sample comprises electrophoresis.
 44. The method of claim 43,wherein the electrophoresis is two-dimensional gel electrophoresis. 45.The method of claim 42, wherein the sample is obtained from mammaliancells or tissue.
 46. The method of claim 45, wherein the mammal is ahuman.
 47. The method of claim 42, wherein transferring of a portion ofthe separated protein comprises gel transfer.
 48. The method of claim42, wherein transferring a portion of the separated protein comprisescontact transfer.
 49. The method of claim 42, wherein the mammaliancells or tissue are human cells or tissue.
 50. The method of claim 42,wherein the separated protein is a product of a human gene.
 51. Themethod of claim 42, wherein at least one of the species of ligand isselected from the group consisting of an antibody, an antibody fragment,a single chain antibody, a receptor protein, a solubilized receptorderivative, a receptor ligands, a metal ion, a virus, a viral protein,an enzyme substrate, a toxin, a toxin candidate, a pharmacologicalagent, and a pharmacological agent candidate.
 52. The method of claim51, wherein at least one of the species of ligand is an antibody or anantibody fragment.
 53. The method of claim 51, wherein at least one ofthe species of ligand is a receptor protein, a solubilized receptorderivative, or a receptor ligand.
 54. The method of claim 51, wherein atleast one of the species of ligand is a pharmacological agent orpharmacological agent candidate.
 55. The method of claim 51, wherein thebinding of at least one of the species of ligand is dependent upon thestructure of the separated protein.
 56. The method of claim 51, whereinthe binding of at least one of the species of ligand is dependent uponthe biological function of the separated protein.
 57. The method ofclaim 42, wherein at least one of the membranes is incubated with morethan one species of ligand.
 58. The method of claim 42, wherein theplurality of membranes comprises at least two membranes.
 59. The methodof claim 58, wherein the plurality of membranes comprises at least 10membranes.
 60. The method of claim 58, wherein the plurality ofmembranes comprises at least 20 membranes.
 61. The method of claim 42,wherein the one or more ligand species comprises at least two ligandspecies.
 62. The method of claim 61, wherein the one or more ligandspecies comprises at least 10 ligand species.
 63. The method of claim61, wherein the one or more ligand species comprises at least 20 ligandspecies.
 64. The method of claim 42, wherein incubating each of themembranes is performed before separating at least one protein.
 65. Amethod for uniquely visualizing a desired predetermined protein ifpresent in a biological sample, the method comprising: separatingproteins present in the sample from one another; transferring a portionof the separated proteins of the sample to a plurality of membranes in astacked configuration; incubating each of the membranes in the presenceof one or more species of predetermined ligand molecules underconditions sufficient to permit binding between desired predeterminedprotein and a ligand capable of binding to such protein; and visualizingany binding between the protein and any of the species of predeterminedligand molecules.
 66. A method for identifying biomolecules that havebeen separated on a solid support, the method comprising: contacting astack of membranes to the solid support containing the separatedbiomolecules; permitting biomolecules to be transferred from the solidsupport to multiple membranes in the stack; separating the membranesfrom the stack; and identifying one or biomolecules transferred to atleast one of the membranes.
 67. The method of claim 66, wherein themethod is a method for identifying proteins, and the biomoleculescomprise proteins.
 68. The method of claim 66, wherein the membraneshave a high affinity but a low capacity for at least one class ofbiomolecule.
 69. The method of claim 67, wherein the membranes have ahigh affinity but a low capacity for proteins.
 70. The method of claim66, wherein at least some of the biomolecules are transferred to eachmembrane of the stack.
 71. The method of claim 66, wherein permittingthe biomolecules to be transferred from the support to multiplemembranes in a stack produces multiple replicate membranes.
 72. Themethod of claim 66, wherein the biomolecules are separated on a gel. 73.The method of claim 72, wherein the separation compriseselectrophoresis.
 74. The method of claim 73, wherein the electrophoresisis SDS PAGE.
 75. The method of claim 74, wherein more than 30 microgramsof protein is loaded into a well of the gel.
 76. The method of claim 75,wherein about 50 to about 100 micrograms of protein are loaded into awell of the gel.
 77. The method of claim 69, wherein the membranescomprise polycarbonate.
 78. The method of claim 69, wherein themembranes comprise a cellulose derivative.
 79. The method of claim 78,wherein the cellulose derivative is cellulose acetate.
 80. The method ofclaim 66, wherein the membranes comprise a polyolefin.
 81. The method ofclaim 66, wherein the stack comprises at least 5 membranes.
 82. Themethod of claim 81, wherein the stack comprises at least 10 membranes.83. The method of claim 66, wherein each membrane is less than about 30microns thick.
 84. The method of claim 83, wherein each membrane isabout 8 to 10 microns thick.
 85. The method of claim 69, wherein atleast one side of the membranes is treated to increase specific bindingof proteins.
 86. The method of claim 85, wherein at least one side ofthe membranes is treated to increase specific binding of the proteins orother targeted proteins.
 87. The method of claim 85, wherein thetreatment comprises a coating of nitrocellulose.
 88. The method of claim85, wherein the treatment comprises a coating of poly-L-lysine.
 89. Themethod of claim 66, wherein the membranes are in a frame, the framebeing mounted to the periphery of the membranes, wherein the framedefines a channel for passing fluids or air away from the spaceintermediate the membrane and an adjacent membrane.
 90. A method foridentifying biomolecules that have been separated on a solid support,the method comprising: providing a solid support containing theseparated biomolecules wherein the support has an upper side and a lowerside; applying a first stack of membranes to the upper side and a secondstack of membranes to the lower side; permitting the biomolecules to betransferred from the support to the first and second membrane stacks;separating the membranes, and identifying one or more biomoleculestransferred to at least one of the membranes.
 91. The method of claim90, wherein the biomolecules comprise proteins.
 92. A kit comprising: amembrane array for detecting biomolecules in a sample, the arraycomprising a plurality of membranes, wherein each of the plurality ofmembranes has substantially a same affinity for the biomolecules; andcontainers of detector molecules for detecting biomolecules captured oneach membrane.
 93. The kit of claim 92, wherein the detector moleculesare antibodies or probes.
 94. The kit according to claim 92 wherein themembranes comprise a polymer substrate coated with a material forincreasing an affinity of the substrate to the biomolecules.
 95. The kitaccording to claim 94 wherein the coating material comprisesnitrocellulose.
 96. The kit according to claim 93, wherein theantibodies or probes are specific capture molecules for biomoleculessought to be detected on particular membranes of the array.
 97. The kitaccording to claim 96 wherein each container contains an antibodycocktail, and each antibody cocktail comprises at least two antibodieswith different binding specificity.
 98. The kit according to claim 92wherein the plurality of membranes has a low capacity for thebiomolecules.
 99. The kit according to claim 92 wherein the plurality ofmembranes each have a thickness of less than about 30 microns.
 100. Thekit according to claim 99, wherein the plurality of membranes comprise acore substrate comprising polycarbonate, and a coating comprisingnitrocellulose.
 101. A kit for comparing the molecular profiles oftissue samples, comprising: at least one tissue microarray; and at leastone replicate of the tissue microarray.
 102. The kit of claim 101,wherein the at least one replicate of the tissue microarray was producedusing the method of claim
 38. 103. A kit for replicating a pattern ofbiomolecules from a tissue sample, comprising: a plurality of membranes,each having a coating on its upper and/or lower surfaces to increasespecific binding of a target biomolecule; a quantity of transfer buffer;and a fluid impervious enclosure.
 104. The kit of claim 103, furthercomprising instructions for carrying out the method of claim
 32. 105. Akit for analyzing a proteome comprising: a plurality of membranes, eachhaving a affinity for at least one protein; and a plurality of reagentspecies, each adapted to detect one or more specific proteins bound tothe membranes.
 106. The kit of claim 105, further comprisinginstructions setting forth the particular groups of reagents to beapplied to each of the membranes.
 107. The kit of claim 104, wherein themembranes comprise a porous substrate having a thickness of less thanabout 30 microns.
 108. The kit of claim 107, wherein the membranes arepolycabonate membranes, coated with a material for increasing theaffinity of the membrane to biomolecules.
 109. The kit of claim 108,wherein the membranes are coated with nitrocellulose.
 110. The kitaccording to claim 105 wherein the reagent species are selected from thegroup consisting of an antibody, an antibody fragment, a single chainantibody, a receptor protein, a solubilized receptor derivative, areceptor ligands, a metal ion, a virus, a viral protein, an enzymesubstrate, a toxin, a toxin candidate, a pharmacological agent, and apharmacological agent candidate.
 111. A kit for uniquely visualizing adesired predetermined protein if present in a biological sample,comprising: a plurality of membranes, each having a specific affinityfor at least one protein, and a plurality of reagent species, eachadapted to detect the desired predetermined protein if bound to themembranes.
 112. A membrane unit for blotting comprising: a stack of atleast two porous membranes having a thickness no greater than about 30microns; and a frame, mounted to the membranes, the frame a having athickness no greater than about 300 microns.
 113. The membrane unit ofclaim 112, wherein the unit comprises more than two membranes.
 114. Themembrane unit of claim 112, wherein the unit comprises a frame, mountedto the periphery of the membranes, wherein the frame defines a channelfor passing fluids or air away from the space intermediate the membraneand an adjacent membrane.
 115. The membrane unit of claim 112, whereinthe membranes have a high affinity but a low capacity for proteins. 116.The membrane unit of claim 115, wherein the membranes comprisepolycarbonate.
 117. The membrane unit of claim 115, wherein themembranes comprise a cellulose derivative.
 118. The membrane unit ofclaim 117, wherein the cellulose derivative is cellulose acetate. 119.The membrane unit of claim 115, wherein the membranes comprise apolyolefin.
 120. The membrane unit of claim 112, wherein the stackcomprises at least 5 membranes.
 121. The membrane unit of claim 120,wherein the stack comprises at least 10 membranes.
 122. The membraneunit of claim 112, wherein the thickness of the membranes is less thanabout 30 microns.
 123. The membrane unit of claim 122, wherein thethickness of the membrane is about 8 to 10 microns.
 124. The membraneunit of claim 112, wherein at least one side of the membranes is treatedto increase specific binding of a biomolecule.
 125. The membrane unit ofclaim 115, wherein at least one side of the membranes is treated toincrease specific binding of the proteins or other targeted proteins.126. The membrane unit of claim 125, wherein the treatment comprises acoating of nitrocellulose.
 127. The membrane unit of claim 115, whereinthe treatment comprises a coating of poly-L-lysine.
 128. A membrane foruse in the membrane unit of claim
 112. 129. The membrane of claim 128,wherein at least one side of the membranes is treated to increasespecific binding of a targeted biomolecule.
 130. A porous membranehaving a high affinity but low capacity for biomolecules, the membranecomprising a core substrate and a coating, wherein the membrane has athickness of no more than about 30 microns.
 131. The membrane of claim130, wherein the core substrate comprises polycarbonate, celluloseacetate, a polyolefin, or combinations of two or more thereof.
 132. Themembrane of claim 130, wherein the coating comprises nitrocellulose,poly-L-lysine, or mixtures thereof.
 133. The membrane of claim 130,wherein the core substrate comprises polycarbonate and the coatingcomprises nitrocellulose.
 134. The membrane of claim 130, wherein themembrane has a thickness of about 8-10 microns.